Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation

ABSTRACT

The present invention provides tumor cell preparations for use as models of the EMT process for use in the identification of anti-cancer agents, wherein said tumor cell preparations comprise cells of the epithelial tumor cell line CFPAC-1, which are stimulated by receptor ligands to induce EMT, or which have been engineered to inducibly express a protein that stimulates EMT. The present invention also provides methods of identifying potential anti-cancer agents by using such tumor cell preparations to identify agents that inhibit EMT, stimulate MET, or inhibit the growth of mesenchymal-like cells. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors, which appear to be less effective at inhibiting tumor cells that have undergone an EMT.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/208,898, filed Feb. 27, 2009, which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention is directed to EMT cell models and methods for their use in the identification of new anti-cancer agents for treating cancer patients, particularly in combination with other agents such as EGFR or IGF-1R kinase inhibitors that can be less effective at inhibiting tumor cells that have undergone an EMT. Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body.

An anti-neoplastic drug would ideally kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess such an ideal profile. Instead, most possess very narrow therapeutic indexes. Furthermore, cancerous cells exposed to slightly sub-lethal concentrations of a chemotherapeutic agent will very often develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents as well.

A multitude of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. The most commonly used types of anticancer agents include: DNA-alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNA intercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormone therapy (e.g., tamoxifen, flutamide). More recently, gene targeted therapies, such as protein-tyrosine kinase inhibitors have increasingly been used in cancer therapy (de Bono J. S. and Rowinsky, E. K. (2002) Trends in Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313). Such approaches, such as the EGFR kinase inhibitor erlotinib, are generally associated with reduced toxicity compared with conventional cytotoxic agents. They are therefore particularly appropriate for use in combination regimens. In pancreatic cancer, phase III trials have shown that first-line erlotinib treatment in combination with gemcitabine improves survival.

The epidermal growth factor receptor (EGFR) family comprises four closely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved in cellular responses such as differentiation and proliferation. Over-expression of the EGFR kinase, or its ligand TGF-alpha, is frequently associated with many cancers, including breast, lung, colorectal, ovarian, renal cell, bladder, head and neck cancers, glioblastomas, and astrocytomas, and is believed to contribute to the malignant growth of these tumors. A specific deletion-mutation in the EGFR gene (EGFRvIII) has also been found to increase cellular tumorigenicity. Activation of EGFR stimulated signaling pathways promote multiple processes that are potentially cancer-promoting, e.g. proliferation, angiogenesis, cell motility and invasion, decreased apoptosis and induction of drug resistance. Increased HER1/EGFR expression is frequently linked to advanced disease, metastases and poor prognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFR expression has been shown to correlate with a high metastatic rate, poor tumor differentiation and increased tumor proliferation.

Mutations which activate the receptor's intrinsic protein tyrosine kinase activity and/or increase downstream signaling have been observed in NSCLC and glioblastoma. However the role of mutations as a principle mechanism in conferring sensitivity to EGF receptor inhibitors, for example erlotinib (TARCEVA®) or gefitinib (IRESSA™), has been controversial. Recently, a mutant form of the full length EGF receptor has been reported to predict responsiveness to the EGF receptor tyrosine kinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science 304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med. 350:2129-2139). Cell culture studies have shown that cell lines which express the mutant form of the EGF receptor (i.e. H3255) were more sensitive to growth inhibition by the EGF receptor tyrosine kinase inhibitor gefitinib, and that much higher concentrations of gefitinib was required to inhibit the tumor cell lines expressing wild type EGF receptor. These observations suggests that specific mutant forms of the EGF receptor may reflect a greater sensitivity to EGF receptor inhibitors, but do not identify a completely non-responsive phenotype.

Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is an orally available inhibitor of EGFR kinase. In vitro, erlotinib has demonstrated substantial inhibitory activity against EGFR kinase in many human tumor cell lines. In a phase III trial, erlotinib monotherapy significantly prolonged survival, delayed disease progression and delayed worsening of lung cancer-related symptoms in patients with advanced, treatment-refractory NSCLC (Shepherd, F. et al. (2005) N. Engl. J. Med. 353(2):123-132). In November 2004 the U.S. Food and Drug Administration (FDA) approved TARCEVA® for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen.

The development for use as anti-tumor agents of compounds that directly inhibit the kinase activity of IGF-1R, as well as antibodies that reduce IGF-1R kinase activity by blocking IGF-1R activation or antisense oligonucleotides that block IGF-1R expression, are also areas of intense research effort (e.g. see Larsson, O. et al (2005) Brit. J. Cancer 92:2097-2101; Ibrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res. 11:944s-950s; Mitsiades, C. S. et al. (2004) Cancer Cell 5:221-230; Camirand, A. et al. (2005) Breast Cancer Research 7:R570-R579 (DOI 10.1186/bcr1028); Camirand, A. and Pollak, M. (2004) Brit. J. Cancer 90:1825-1829; Garcia-Echeverria, C. et al. (2004) Cancer Cell 5:231-239).

IGF-1R is a transmembrane RTK that binds primarily to IGF-1 but also to IGF-II and insulin with lower affinity. Binding of IGF-1 to its receptor results in receptor oligomerization, activation of tyrosine kinase, intermolecular receptor autophosphorylation and phosphorylation of cellular substrates (major substrates are IRS1 and Shc). The ligand-activated IGF-1R induces mitogenic activity in normal cells and plays an important role in abnormal growth. A major physiological role of the IGF-1 system is the promotion of normal growth and regeneration. Overexpressed IGF-1R (type 1 insulin-like growth factor receptor) can initiate mitogenesis and promote ligand-dependent neoplastic transformation. Furthermore, IGF-1R plays an important role in the establishment and maintenance of the malignant phenotype. Unlike the epidermal growth factor (EGF) receptor, no mutant oncogenic forms of the IGF-1R have been identified. However, several oncogenes have been demonstrated to affect IGF-1 and IGF-1R expression. The correlation between a reduction of IGF-1R expression and resistance to transformation has been seen. Exposure of cells to the mRNA antisense to IGF-1R RNA prevents soft agar growth of several human tumor cell lines. IGF-1R abrogates progression into apoptosis, both in vivo and in vitro. It has also been shown that a decrease in the level of IGF-1R below wild-type levels causes apoptosis of tumor cells in vivo. The ability of IGF-1R disruption to cause apoptosis appears to be diminished in normal, non-tumorigenic cells.

The IGF-1 pathway in human tumor development has an important role. IGF-1R overexpression is frequently found in various tumors (breast, colon, lung, sarcoma) and is often associated with an aggressive phenotype. High circulating IGF1 concentrations are strongly correlated with prostate, lung and breast cancer risk. Furthermore, IGF-1R is required for establishment and maintenance of the transformed phenotype in vitro and in vivo (Baserga R. Exp. Cell. Res., 1999, 253, 1-6). The kinase activity of IGF-1R is essential for the transforming activity of several oncogenes: EGFR, PDGFR, SV40 T antigen, activated Ras, Raf, and v-Src. The expression of IGF-1R in normal fibroblasts induces neoplastic phenotypes, which can then form tumors in vivo. IGF-1R expression plays an important role in anchorage-independent growth. IGF-1R has also been shown to protect cells from chemotherapy-, radiation-, and cytokine-induced apoptosis. Conversely, inhibition of endogenous IGF-1R by dominant negative IGF-1R, triple helix formation or antisense expression vector has been shown to repress transforming activity in vitro and tumor growth in animal models.

During most cancer metastases, an important change occurs in a tumor cell known as the epithelial-mesenchymal transition (EMT) (Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515)). EMT does not occur in healthy cells except during embryogenesis. Epithelial cells, which are bound together tightly and exhibit polarity, give rise to mesenchymal cells, which are held together more loosely, exhibit a loss of polarity, and have the ability to travel. These mesenchymal cells can spread into tissues surrounding the original tumor, as well as separate from the tumor, invade blood and lymph vessels, and travel to new locations where they divide and form additional tumors. Recent research has demonstrated that epithelial cells respond well to EGFR and IGF-1R kinase inhibitors, but that after an EMT the resulting mesenchymal-like cells are much less sensitive to such inhibitors. (e.g. Thompson, S. et al. (2005) Cancer Res. 65(20):9455-9462; U.S. Patent Application 60/997,514). Thus there is a pressing need for anti-cancer agents that can prevent or reverse tumor cell EMT events (e.g. stimulate a mesenchymal to epithelial transition (MET)), or inhibit the growth of the mesenchymal-like tumor cells resulting from EMT. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors.

As human cancers progress to a more invasive, metastatic state, multiple signaling programs regulating cell survival and migration programs are observed depending on cell and tissue contexts (Gupta, G. P., and Massague, J. (2006) Cell 127, 679-695). Recent data highlight the transdifferentiation of epithelial cancer cells to a more mesenchymal-like state, a process resembling epithelial-mesenchymal transition (EMT; (Oft, M., et al. (1996). Genes & development 10, 2462-2477; Perl, A. K., et al. (1998). Nature 392, 190-193), to facilitate cell invasion and metastasis (Brabletz, T. et al. (2005) Nat Rev Cancer 5, 744-749; Christofori, G. (2006) Nature 441, 444-450). Through EMT-like transitions mesenchymal-like tumor cells are thought to gain migratory capacity at the expense of proliferative potential. A mesenchymal-epithelial transition (MET) has been postulated to regenerate a more proliferative state and allow macrometastases resembling the primary tumor to form at distant sites (Thiery, J. P. (2002) Nat Rev Cancer 2, 442-454). EMT-like transitions in tumor cells result from transcriptional reprogramming over considerable periods of time (weeks to months) via transcription factors harboring zinc finger, forkhead, bHLH and HMG-box domains (Mani, S. A. et al. (2007) Proceedings of the National Academy of Sciences of the United States of America 104, 10069-10074; Peinado, H. et al. (2007) Nat Rev Cancer 7, 415-428). The loss of E-cadherin and transition to a more mesenchymal-like state likely serves a major role in the progression of cancer (Matsumura, T. et al. (2001) Clin Cancer Res 7, 594-599; Yoshiura, K. et al. (1995). Proceedings of the National Academy of Sciences of the United States of America 92, 7416-7419) and the acquisition of a mesenchymal phenotype has been correlated with poor prognosis (Baumgart, E. et al. (2007) Clin Cancer Res 13, 1685-1694; Kokkinos, M. I. Et al. (2007) Cells, tissues, organs 185, 191-203; Willipinski-Stapelfeldt, B. et al. (2005) Clin Cancer Res 11, 8006-8014.). Targeting tumor-derived and/or tumor-associated stromal cells provides a unique mechanism to block EMT-like transitions and inhibit the survival of invading cells.

The cellular changes associated with EMT-like transitions alter the dependence of carcinoma cells on EGF receptor signaling networks for survival. It has been observed that an EMT-like transition was associated with cellular insensitivity to the EGFR-TKI erlotinib (Thomson, S. et al. (2005) Cancer research 65, 9455-9462; Witta, S. E., et al. (2006) Cancer research 66, 944-950; Yauch, R. L., et al. (2005) Clin Cancer Res 11, 8686-8698), in part from EGFR independent activation of either or both the PI3′ kinase or Mek-Erk pathways (Buck, E. et al. (2007). Molecular cancer therapeutics 6, 532-541). Similar data correlating EMT status to sensitivity to EGFR TKIs have been reported in pancreatic, CRC (Buck, E. et al. (2007) Molecular cancer therapeutics 6, 532-541) bladder (Shrader, M. et al. (2007) Molecular cancer therapeutics 6, 277-285) and HNSCC (Frederick et al. (2007) Molecular cancer therapeutics 6, 1683-1691) cell lines, xenografts and in patients (Yauch, R. L., et al. (2005) Clin Cancer Res 11, 8686-8698). The molecular determinants to alternative routes of activation of the PI3′ kinase and Erk pathways, which can bypass cellular sensitivity to EGF receptor inhibitors, have been actively investigated (Chakravarti, A. et al. (2002) Cancer research 62, 200-207; Engelman, J. A. et al. (2007) Science 316:1039-1043).

Inhibition of EMT-like transitions and mesenchymal-like cell survival would be predicted to reduce tumor metastasis and progression. Current data suggest patients with metastasis have heterogeneous tumors that can contain cells with epithelial and mesenchymal-like phenotypes. The observation that tumors can acquire new signaling pathways, for example PDGFR and FGFR autocrine signaling, suggest new therapeutic modalities to target specific tumor cell populations. These data suggest rational drug combinations that would not only cause growth inhibition or apoptosis of tumor cells directly, but would also impact mesenchymal cell populations promoting cancer recurrence (Moody, S. E. et al. (2005). Cancer cell 8, 197-209). Essential for the discovery and development of such drug combinations will be the availability of good cellular and animal models where their efficacy can be readily assessed. The invention described herein provides such models.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a test agent to be screened, contacting the sample with a single protein ligand preparation that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agent that inhibits tumor cells that have undergone an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a mesenchymal-like tumor cell preparation for use in the identification of anti-cancer agents, wherein said tumor cell preparation is prepared by a process comprising: contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a tumor cell preparation for use in the identification of anti-cancer agents, wherein said tumor cell preparation comprises: a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells. The protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells may be Snail, Zeb-1, HGF or OSM. The present invention also provides methods of identifying potential anti-cancer agents by using such tumor cell preparations to identify agents that inhibit EMT, stimulate MET, or inhibit the growth of mesenchymal-like cells.

The present invention also provides methods for preparing compositions comprising agents identified by any of the methods described herein, to be used in the treatment of tumors or tumor metastases.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1: Induction of EMT in CFPAC-1 cells by growth factors and cytokines. CFPAC-1 cells were treated with growth factors or cytokines for 7 days (see Materials and Methods), and protein biomarkers of EMT, E-cadherin and vimentin measured by immunoblotting of cell extracts. Factors included: SDF-1a (stromal derived factor-1), TRANCE (TNF-related activation induced cytokine), FGF1 (fibroblast growth factor acidic), FGF2 (fibroblast growth factor basic), BMP4 (bone morphogenetic protein 4), BMP7 (bone morphogenetic protein 7), IGF2 (insulin-like growth factor 2), OSM (Oncostatin M), NRG1 (neuregulin-1), PDGFaa (platelet derived growth factor a), PDGFbb (platelet derive growth factor b), Ang2 (angiopoeitin 2), amphiregulin, HMGB 1 (Human high-mobility group box 1 protein), LIF (Leukemia inhibitory factor), GM-CSF (Granulocyte macrophage colony stimulating factor), M-CSF (Macrophage colony stimulating factor), PAR1 (Protease activated receptor 1 agonist), PAR2 (Protease activated receptor 2 agonist), PAR4 (Protease activated receptor 4 agonist), HGF (Hepatocyte Growth Factor), IL-31 (Interleukin-31), IL-33 (Interleukin-33), IL 1-a (Interleukin-1 alpha), CTGF (Connective tissue growth factor), WISP1 (Wnt-1 inducible signaling pathway protein), PTHrP (Polypeptide hormone-related protein), MCP1 (monocyte chemotactic protein-1), and Fulvestrant at 1 μM.

FIG. 2: Titration of HGF and OSM treatment of CFPAC1 cells. Varying concentrations of HGF or OSM was used and the protein biomarkers were analysed by immunoblotting analysis of E-cadherin and vimentin in lysates of CFPAC-1 cells.

FIG. 3: CFPAC-1 morphology changes with HGF and OSM induction of EMT. CFPAC-1 cells were treated with HGF or OSM for 7 days and cell morphology was determined by light microscopy. Control untreated CFPAC1 cells formed colonies while HGF and OSM treated cells have were less compact with more elongated cell morphology.

FIG. 4: Confocal images of EMT induced changes in CFPAC-1 cells. CFPAC-1 cells were treated with HGF, OSM or combination of HGF and OSM for 7 days and stained for localization of E-cadherin (green) and vimentin (red). Absence of E-cadherin expression or mislocalization of E-cadherin to the cytoplasm and more pronounced staining of vimentin was observed with growth factor or cytokine treatment as a result of EMT.

FIG. 5: Changes in migration and invasion of CFPAC-1 cells induced to undergo EMT. CFPAC-1 cells were treated with HGF, OSM or the combination of HGF and OSM for 7 days. (A) CFPAC-1 cells were monitored for their capacity to migrate in response to FBS with HGF (p value=0.04), OSM (p value <0.0001) and HGF+OSM (p value <0.0001). (B) CFPAC-1 cells were monitored for their capacity to invade through collagen I in response to FBS (p value of <0.0001 for all treatments). (C)CFPAC-1 cells were monitored for their capacity to invade through BME (basement membrane extract) in response to FBS (HGF, p value=0.03, OSM p value=0.002, HGF+OSM, p value=0.003).

FIG. 6: EMT induced in CFPAC-1 cells can be reversed resulting in a mesenchymal to epithelial transition (MET). (A) CFPAC-1 cells were treated with HGF, OSM or HGF+OSM for 14 days (D14). Ligand was removed and monitored for ability of cells to revert to a more epithelial morphology. Cells were monitored on day 3 (D17), day 7 (D21) and day 12 (D26) after ligand removal. Cells reverted to a more epithelial morphology with complete reversion with 12 days of ligand withdrawal. (B) Immunoblotting analysis was performed with CFPAC cell lysates corresponding to samples in panel A. E-cadherin is prominent beginning on day 3 of ligand withdrawal (labeled Day 17) and complete absence of vimentin is observed after 12 days of ligand withdrawal (labeled Day 26).

FIG. 7: Induction of EMT in CFPAC-1 with OSM can be inhibited with a JAK small molecule inhibitor. CFPAC-1 cells treated for 7 days with HGF or OSM in presence or absence of JAK small molecule inhibitor (0.25 μM) or MEK small molecule inhibitor (3 μM). JAK inhibitor blocked the morphology change when CFPAC-1 cells were treated with OSM (Top panel of light microscopy). Protein biomarkers were analysed by immunoblotting analysis of E-cadherin and vimentin.

FIG. 8: Immunofluorescence of E-cadherin and vimentin in CFPAC1 cells. Cells were treated for 7 days with the indicated ligands and stained for E-cadherin and vimentin as described. All treatments result in downregulation of E-cadherin, loss of cell-cell contacts and upregulation of vimentin.

FIG. 9: Impact of EMT on 3D growth. CFPAC1 cells were grown in Matrigel for 14 days with or without HGF+OSM. Cells were then fixed and stained for E-cadherin and vimentin with TOPRO3 as a nuclear counterstain. EMT correlates with downregulation of E-cadherin expression, an increase in vimentin expression and a decreased organization of colony architecture.

FIG. 10: EMT characteristics of CFPAC1 model in vivo. (A) Western Blotting and PCR of E-cadherin and vimentin. CFPAC1 cells were implanted orthotopically in nude mice and allowed to grow for 2, 4 or 8 weeks before harvesting. Protein lysates were prepared from tumors and immunoblotted for E-Cadherin and vimentin. The western blot shows 4 individual tumors, and the PCR result is mean fold change in mRNA between 4 tumors from week 2 to week 8. (B) Immunohistochemistry of E-cadherin and vimentin in CFPAC in a representative orthotopic 8 week tumor. Tumors sections were stained for E-cadherin and vimentin.

DETAILED DESCRIPTION OF THE INVENTION

The term “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.

“Cell growth”, as used herein, for example in the context of “tumor cell growth”, unless otherwise indicated, is used as commonly used in oncology, where the term is principally associated with growth in cell numbers, which occurs by means of cell reproduction (i.e. proliferation) when the rate the latter is greater than the rate of cell death (e.g. by apoptosis or necrosis), to produce an increase in the size of a population of cells, although a small component of that growth may in certain circumstances be due also to an increase in cell size or cytoplasmic volume of individual cells. An agent that inhibits cell growth can thus do so by either inhibiting proliferation or stimulating cell death, or both, such that the equilibrium between these two opposing processes is altered.

“Tumor growth” or “tumor metastases growth”, as used herein, unless otherwise indicated, is used as commonly used in oncology, where the term is principally associated with an increased mass or volume of the tumor or tumor metastases, primarily as a result of tumor cell growth.

“Abnormal cell growth”, as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes the abnormal growth of: (1) tumor cells (tumors) that proliferate by expressing a mutated tyrosine kinase or over-expression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (4) any tumors that proliferate by receptor tyrosine kinases; (5) any tumors that proliferate by aberrant serine/threonine kinase activation; and (6) benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.

The term “treating” as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient with cancer. The term “treatment” as used herein, unless otherwise indicated, refers to the act of treating.

The phrase “a method of treating” or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in an animal, or to alleviate the symptoms of a cancer. “A method of treating” cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an animal, is nevertheless deemed an overall beneficial course of action.

The term “therapeutically effective agent” means a composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The term “therapeutically effective amount” or “effective amount” means the amount of the subject compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The present invention derives from research that provided methods for determining which tumors will respond most effectively to treatment with protein-tyrosine kinase inhibitors (e.g. Thompson, S. et al. (2005) Cancer Res. 65(20):9455-9462; U.S. Patent Application 60/997,514) based on whether the tumor cells have undergone an epithelial to mesenchymal transition (“EMT”; Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515). This research demonstrated that epithelial cells respond well to EGFR and IGF-1R kinase inhibitors, but that after an EMT the resulting mesenchymal-like cells are much less sensitive to such inhibitors. Biomarkers can be used to determine whether tumor cells have undergone an EMT (Thomson, S. et al. (2005) Cancer Res. 65(20):9455-9462). As a result of such work it became apparent that new therapeutic approaches would be necessary to find agents that were capable of inhibiting the genesis, growth and/or function of such mesenchymal-like cells, which are thought to be an important element in the invasive and metastatic properties of tumors.

A considerable body of work is emerging that is beginning to delineate the biochemical pathways involved in regulating tumor EMT events, and to characterize the resultant mesenchymal-like tumor cells. For example, experiments using specific siRNA inhibitors of the expression of various protein products produced by mesenchymal-like tumor cells have demonstrated that reduced expression of the products of certain genes can specifically inhibit the growth of mesenchymal-like tumor cells. Thus pharmacological agents that also specifically inhibit the expression of the protein products encoded by these genes, or specifically inhibit the biological activity of the expressed proteins (e.g. phosphotransferase activity), such as specific antibodies to expressed proteins that possess an extracellular domain, antisense molecules, ribozymes, or small molecule enzyme inhibitors (e.g. protein kinase inhibitors), are similarly expected to be agents that will also specifically inhibit the growth of mesenchymal-like tumor cells. The anti-tumor effects of a combination of an EGFR or IGF-1R kinase inhibitor with such an agent should be superior to the anti-tumor effects of these kinase inhibitors by themselves, since such a combination should effectively inhibit both epithelial and mesenchymal-like tumor cells, and thus co-administration of such agents with EGFR or IGF-1R kinase inhibitors should be effective for treatment of patients with advanced cancers such as NSCL, pancreatic, colon or breast cancers.

Given the identification of key targets for the discovery and development of agents that will inhibit the growth of mesenchymal-like tumor cells, or the EMT process, there is thus a pressing need for models and methods to evaluate agents identified by in vitro screening methods (e.g. using protein kinase assays) to determine if they have the predicted effect of inhibiting the growth and/or migration of mesenchymal-like tumor cells, both at a cellular level, and in vivo. The data presented in the Examples herein demonstrates that the epithelial tumor cell line CFPAC-1 can be stimulated to undergo EMT by contacting it with one or more cell receptor ligands, or by inducing the expression of one or more protein products capable of modulating the EMT process. Not all epithelial tumor cell lines can be stimulated to undergo EMT, and the optimal process for EMT induction is cell line dependent. Thus, CFPAC-1 tumor cells, treated in various ways to induce EMT, can be used as models, both in vitro and vivo, in methods to identify agents that can inhibit mesenchymal-like tumor cells, prevent their formation, or reverse the EMT process. These methods are also useful in the identification of agents for the treatment of fibrotic disorders resulting in part from EMT transitions, including but not limited to renal fibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. Thus any of the inventions described herein as being applicable to tumor cells, will also be applicable to other cell types involved in fibrotic diseases that undergo EMT. Similarly, any of the inventions described herein as being useful for the identification of anti-cancer agents, will also be useful in the identification of anti-fibrotic agents for treating diseases that involve fibrosis.

“CFPAC-1 cells” as used herein, refers to cells of the pancreatic epithelial cell line CFPAC-1 [a.k.a. CRL-1918™] available from the American Tissue Culture Collection (ATCC) as ATCC® Number:CRL-1918™, derived from a pancreatic ductal adenocarcinoma (liver metastasis) from a patient with cystic fibrosis.

Accordingly, the present invention provides a method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a test agent to be screened, contacting the sample with a single protein ligand preparation that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

A “single protein ligand preparation” as used herein, means a preparation comprising a protein ligand for a cell receptor, which ligand is capable, by itself, of substantially inducing EMT in CFPAC-1 cells, as assessed for example by a significant decrease in expression of the epithelial biomarker E-cadherin, and/or a significant increase in expression of the mesenchymal biomarker vimentin. A single protein ligand preparation may contain additional compounds or proteins, e.g. cell nutrients, other growth factors, agents that stabilize the protein ligands, etc. For example, a single protein ligand preparation may be supplemented with one or more alternative single ligands that also induce EMT, or a ligand that does not normally induce EMT on its own, which may result in a slightly more complete EMT. By contrast with CFPAC-1 cells, induction of EMT in other cells can sometimes require a “dual protein ligand preparation”, i.e. a preparation comprising two protein ligands for different cell receptors, which ligands are capable of inducing EMT in cells, and where both ligands are required for EMT induction (i.e. either ligand by itself does not substantially induce EMT) (e.g. EMT induction in H358 cells by HGF and OSM, see U.S. provisional application 61/068,612).

In any of the methods or cell preparations of the invention described herein, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells may be selected from any of the protein ligands that bind to and activate the receptors for OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4, either presently known, or yet to be discovered or synthesized. For example, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells may be selected from OSM (Oncostatin M; NCIB GeneID: 5008); HGF (hepatocyte growth factor; NCIB GeneID: 3082); BMP7 (bone morphogenetic protein 7; NCIB GeneID: 655); IGF2 (insulin-like growth factor 2 (a.k.a. somatomedin A); NCIB GeneID: 3481); LIF (Leukemia inhibitory factor; NCIB GeneID: 3976); PAR2 agonists, e.g. SLIGKV-NH₂; IL-33 (Interleukin-33; NCIB GeneID: 90865); PAR4 agonists, e.g. AYPGKF-NH₂; CTGF (Connective tissue growth factor; NCIB GeneID: 1490); and BMP4 (bone morphogenetic protein 4; NCIB GeneID: 652). In a preferred 8embodiment the single protein ligand is HGF or OSM; either optionally supplemented with one of BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4. In an alternative embodiment, the single protein ligand is a ligand selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4; optionally supplemented with one of OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4. The human versions of the above ligand proteins are preferred, but where an alternative animal version exists (e.g. from mouse, rat, rabbit, dog, monkey, pig, etc) that also has activity in stimulating the human receptor on CFPAC-1 cells, and induces EMT, this may also be used.

In any of the methods or cell preparations of the invention described herein, when a more complete EMT is sought, as judged by E-cadherin or vimentin expression, the preferred single protein ligands to induce an epithelial-to-mesenchymal transition in CFPAC-1 cells may comprise a ligand that binds to and activates the oncostatin-M receptor (e.g. OSM) or a ligand that binds to and activates the HGF receptor (a.k.a. Met receptor tyrosine kinase) (e.g. HGF). In an alternative embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells may comprise a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of oncostatin M to its receptor (e.g. a ligand that binds to and activates the oncostatin-M receptor (e.g. oncostatin M), or a protein ligand that binds to another receptor that activates the same signal transduction pathways as are activated by binding of oncostatin M to its receptor (i.e. JAK-STAT pathways)); or a ligand that binds to a cell tyrosine kinase receptor and activates the same signal transduction pathways that are activated by the binding of HGF to its receptor (i.e. the PI3K and MAPK pathways; as activated by binding of HGF to Met receptor tyrosine kinase).

Receptor tyrosine kinases that activate the PI3K and MAPK pathways include for example, IGF1-R, FGFR1, FGFR2, FGFR3, FGFR4, heterodimers of FGF receptors 1-4, RON, EGFR, HER-4, heterodimers of HER receptors 1-4, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), and PDGFR (α and β receptor homo- and herero-dimers). Thus, examples of additional ligands that may induce an epithelial-to-mesenchymal transition in CFPAC-1 cells include IGF-1, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF10, macrophage-stimulating protein (MSP; RON receptor ligand), transforming growth factor-α (TGF-α), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AREG), betacellulin (BTC), epiregulin (EREG), epigen (EPGN), neuregulins (NRG-1 (Heregulin), NRG-2, NRG-3, NRG-4), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD, where sufficient receptor levels are present. The human versions of the above ligand proteins are preferred, but where an alternative animal version exists (e.g. from mouse, rat, rabbit, dog, monkey, pig, etc) that also has activity in stimulating the human receptor on CFPAC-1 cells, and induces EMT, this may also be used.

The induction of EMT in CFPAC-1 cells by the specific ligands disclosed herein allows targeting of specific pathways that induce EMT, and thus for identification of anticancer agents that may have different modes of action, and may thus act together in a synergistic manner.

The NCBI GeneID numbers listed herein are unique identifiers of the gene from the NCBI Entrez Gene database record (National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, 8600 Rockville Pike, Building 38A, Bethesda, Md. 20894; Internet address http://www.ncbi.nlm.nih.gov/). They are used herein to unambiguously identify gene products that are referred to elsewhere in the application by names and/or acronyms. Proteins expressed by genes thus identified represent proteins that may be used in the methods of this invention, and the sequences of these proteins, including different isoforms, as disclosed in NCBI database (e.g. GENBANK®) records are herein incorporated by reference.

The sample of cells of the epithelial tumor cell line CFPAC-1 in any of the methods or preparations of this invention can be for example cells in monolayer culture (e.g. cells in a tissue culture plate or dish, e.g. a 96-well plate); cells in three-dimensional culture, such as for example Matrigel™ 3D culture, spheroid cultures, or soft agar culture (Kim, J. B., (2005) Seminars in Cancer Biology 15:365-377; Sutherland, R. M., (1988) Science 240:177-184; Hamilton, G. (1998) Cancer Letters, 131:29-34; or cells in vivo, e.g. a tumor xenograft. In methods described herein that require “an identical sample of cells”, this refers to a sample of cells with essentially the same number of cells, growing under the same conditions. For example, an identical tissue culture dish with approximately the same number of cells, or a tumor xenograft of the same or similar size.

Many biomarkers are known whose level of expression or activity is indicative of the EMT status of tumor cells (e.g. see US Patent Application Publication 2007/0212738; U.S. Patent Application 60/923,463; U.S. Patent Application 60/997,514). Such markers tend to be classified as epithelial or mesenchymal, due to their characteristic association with the particular stage of EMT. Characteristic biomarkers can be, for example, proteins, encoding mRNAs, activity of a gene promoter, level of a transcriptional repressor, or promoter methylation. In any of the methods described herein the biomarker whose expression level is indicative of the EMT status of the sample tumor cells can be an epithelial cell biomarker. Epithelial cell biomarkers include for example E-cadherin, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3. Additional epithelial cell biomarkers include Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, connexin 31, plakophilin 3, stratifin 1, laminin alpha-5, and ST14. In any of the methods described herein the biomarker whose expression level is indicative of the EMT status of the sample tumor cells can also be a mesenchymal cell biomarker. Mesenchymal cell biomarkers include for example is vimentin, fibronectin, N-cadherin, zeb 1, twist, FOXC2 or snail. Additional mesenchymal cell biomarkers include, fibrillin-1, fibrillin-2, collagen alpha2(IV), collagen alpha2(V), LOXL1, nidogen, C11orf9, tenascin, tubulin alpha-3, and epimorphin. Additionally any other epithelial or mesenchymal cell biomarkers known in the art, described herein, or yet to be described, may be used in the methods of the invention described herein. In any of the methods described herein, multiple biomarker level determinations can also be used to assess EMT status, potentially providing a more reliable assessment. For example, an epithelial and a mesenchymal biomarker level may be assessed, the reciprocal changes in each providing internal confirmation that EMT has occurred (e.g. suitable biomarker pairs include for example, E-cadherin/vimentin). In an alternative embodiment, the epithelial biomarker comprises one or more keratins selected from the epithelial keratins 1-28 and 71-80, and the mesenchymal biomarker is vimentin, wherein co-expression of epithelial and mesenchymal biomarkers at similar levels is indicative of a mesenchymal-like tumor cell (see U.S. Patent Application 60/923,463). When used in any of the methods of the invention described herein, epithelial keratins 1-28 and 71-80 includes all the keratins listed in Table 2 herein. In one embodiment of the latter method the epithelial keratin(s) are assessed using a method that will detect all or the majority (i.e. 50% or more) of the keratin biomarkers expressed by the tumor cell (e.g. by using a multi- or pan-specific antibody). In another embodiment of above methods where epithelial keratin biomarker levels are determined, the biomarker comprises keratin 8 and/or keratin 18.

The term “co-expression of epithelial and mesenchymal biomarkers at similar levels” as used herein in the context of determining co-expression of epithelial keratins and the mesenchymal biomarker vimentin means that the ratio of mesenchymal to epithelial biomarker levels is in the range of about 10:1 to about 1:10 (assuming that each biomarker is assayed under comparable conditions, e.g. using antibodies of identical affinity, nucleic acid probes of identical length, identical detection methods, etc.).

In an alternative embodiment of any of the methods described herein that include a step of determining the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells, the biomarker can be the activity of a gene promoter that is altered when the tumor cells undergo EMT. Such promoter activity is readily assessed by incorporating a promoter-reporter construct into the tumor cells and measuring reporter activity. In one embodiment, the activity of an epithelial biomarker gene promoter is assessed by inclusion of an epithelial biomarker gene promoter-reporter gene construct into the CFPAC-1 cells such that said promoter reporter activity can be monitored by reporter gene expression level or activity. For example, the epithelial biomarker gene promoter-reporter gene construct may be an E-cadherin promoter-firefly luciferase construct. In an alternative embodiment, the activity of a mesenchymal biomarker gene promoter is assessed by inclusion of a mesenchymal biomarker gene promoter-reporter gene construct into the CFPAC-1 cells such that said promoter reporter activity can be monitored by reporter gene expression level or activity. For example, the mesenchymal biomarker gene promoter-reporter gene construct may be an vimentin promoter-firefly luciferase construct. The promoter-reporter gene construct may be permanently incorporated into the CFPAC-1 cells as a stable engineered cell line, or may be transiently expressed, using any of the standard techniques for transferring nucleic acid constructs into cells (e.g. transfection, electroporation). Multiple promoter-reporter gene constructs may also be employed in order to monitor several biomarkers simultaneously, e.g. an E-cadherin promoter-firefly luciferase construct and a vimentin promoter-renilla luciferase construct, in order to, for example, monitor simultaneous repression of the E-cadherin gene and induction of the vimentin gene as tumor cells undergo EMT.

The present invention also provides a method of identifying an agent that inhibits tumor cells that have undergone an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4. An alternative embodiment of this method comprises, after the step of determining whether the test agent inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, the additional steps of determining whether an agent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, also inhibits epithelial CFPAC-1 tumor cell growth, and thus determining whether it is an agent that specifically inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition. In an embodiment of the above methods, an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition is determined to do so by stimulating apoptosis of said tumor cells. In another embodiment of the above methods, an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition is determined to do so by inhibiting proliferation of said tumor cells.

The present invention also provides a method of identifying an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a test agent to be screened, contacting the sample with a single protein ligand preparation that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, and admixing the test agent so identified with a carrier, thereby preparing said composition.

The present invention also provides a method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, and admixing the test agent so identified with a carrier, thereby preparing said composition.

The present invention also provides a method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, and admixing the test agent so identified with a carrier, thereby preparing said composition.

The present invention also provides a mesenchymal-like tumor cell preparation for use in the identification of anti-cancer agents, wherein said tumor cell preparation is prepared by a process comprising: contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

For any of the methods described herein, a test agent can be any chemical compound, including small molecules (<approx. 5000 Daltons molecular weight) and macromolecules (e.g. a polypeptide or protein, nucleic acid, glycoprotein, complex carbohydrate, synthetic or natural polymer etc.). Thus, a test agent may be selected from, for example, combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonucleotides and natural product libraries, aptamers, and other entities such as display (e.g. phage display libraries) and antibody products.

The present invention also provides a method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising: contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a test agent to be screened, wherein the cells are implanted orthotopically into the pancreas of an immune-deficient animal, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition. In one embodiment of this method the immune-deficient animal is a nude mouse. In another embodiment, the biomarker whose level is indicative of the EMT status of the sample tumor cells is an epithelial cell biomarker. The epithelial cell biomarker may be, for example, E-cadherin, CDH1 promoter activity, cytokeratin 8, cytokeratin 18, P-cadherin, or erbB3. In another embodiment, the biomarker whose level is indicative of the EMT status of the sample tumor cells is a mesenchymal cell biomarker. The mesenchymal cell biomarker may be, for example, vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb1, twist, FOXC2 or snail.

The present invention also provides an animal model for use in the identification of anti-cancer agents, which comprises a sample of cells of the epithelial tumor cell line CFPAC-1 which have been implanted orthotopically into the pancreas of an immuno-deficient animal. In one embodiment, the immune-deficient animal is a nude mouse (also known as a Foxn1nu mouse).

The present invention also provides a method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a test agent to be screened, contacting the sample with a compound that induces the expression of said protein that stimulates an epithelial to mesenchymal transition in the engineered CFPAC-1 cells, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of engineered CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition.

“Inducibly express”, as used herein, when referring for example to cells which have been engineered to “inducibly express” a protein, means that the protein expression is only turned on by the presence (or absence) of an inducing agent that controls transcription of the gene encoding the protein, which will preferably be incorporated into the cells by stable transformation with a construct containing the gene for the encoding protein under the control of a promoter that is responsive to the inducing agent (i.e. the gene encoding the protein is operably linked to a nucleotide sequence regulating the gene expression, which nucleotide sequence comprises a promoter sequence whose activity can be controlled by the presence of an inducing agent). One example of such an inducible promoter is a tetracycline (tet)-responsive promoter (e.g. a Tet-on system; e.g. see Gossen, M. et al. (1995) Science 268:1766-1769). Such inducible gene expression systems for controlling the expression levels of specific genes of interest are well known in the art (e.g. see Blau, H. M. and Rossi, F. M. V. (1999) Proc. Natl. Acad. Sci. USA 96:797-799; Yamamoto, A. et al. (2001) Neurobiology of Disease 8:923-932; Clackson, T. (2000) Gene Therapy 7:120-125).

For any of the methods or cell preparations described herein involving the epithelial tumor cell line CFPAC-1 which has been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, in one embodiment the cells also comprise a promoter-reporter gene construct that can be similarly inducibly expressed, such that said promoter reporter activity can be monitored by reporter gene expression level or activity, and thus be used to readily assess whether induction of the protein that stimulates an epithelial to mesenchymal transition has been successful. In one example of such an embodiment, the reporter gene is the gene for firefly or Renilla luciferase. Assessment of reporter levels can for example be used for readily monitoring the extent of EMT induction of CFPAC-1 cells, and the location of cells that have undergone EMT. Thus, for example, cells in vivo that have migrated from a primary tumor, or metastasized, can be readily tracked by monitoring such a reporter gene. Thus, the present invention provides a method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition (and metastasis), comprising contacting in vivo a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express both a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, and a reporter gene product (e.g. firefly luciferase), and which have been allowed to form a tumor xenograft in an immunocompromised animal, with a test agent to be screened, contacting the sample with a compound that induces the expression of said protein that stimulates an epithelial to mesenchymal transition in the engineered CFPAC-1 cells and the reporter gene product, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition (and metastasis), by comparing the extent of migration of the sample tumor cells away from a primary tumor (e.g. by imaging analysis of the reporter gene product) to the extent of migration of an identical sample of engineered CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition (and metastasis). Cells derived form an EMT-transition in vivo can also be detected by surgical isolation and immunohistochemistry or in situ hybridization using for example biomarkers as described herein, or in US Patent Application Publication 2007/0212738, U.S. Patent Application 60/923,463, U.S. Patent Application 61/068,612, or U.S. Patent Application 60/997,514).

In one embodiment of the above methods the protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells is Snail. In another embodiment of this method the protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells is Zeb-1. In another embodiment of this method the protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells is OSM; HGF; BMP7; IGF2; LIF; a PAR2 agonist; IL-33; a PAR4 agonist; CTGF; or BMP4. In an alternative embodiment, the protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells may be co-expressed with one or more other proteins that enhance the EMT. Additional EMT causing genes that encode proteins that may be inducibly expressed in engineered CFPAC-1 cells to promote EMT include, but are not limited to, constitutively active cMET receptor; and activated Src kinase (e.g. v-Src or Src Y530F mutants). Similarly, in any of the other methods or cell preparations described herein involving a protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, the protein can be any of the examples described above.

In one embodiment of these methods, a Tet-regulated promoter is used to inducibly express the protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, e.g. a Tet-on system. In one embodiment of this method, the compound that induces the expression of the protein that stimulates an epithelial to mesenchymal transition in the engineered CFPAC-1 cells is doxycycline. Other inducers that may be used include, but are not limited to, tetracycline and anhydrotetracycline. Similarly, in any of the other methods or cell preparations described herein involving a protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, the promoter and inducer compound used to inducibly express the protein can be any of the examples described above. Induction systems include but are not limited to tetracycline regulated plasmids, e.g. Tet-on and Tet-off systems?

In the above methods the biomarker whose level is indicative of the EMT status of the sample tumor cells is for example an epithelial cell biomarker, e.g. E-cadherin, cytoketatin 8, cytokeratin 18, P-cadherin or erbB3, or the activity of an epithelial biomarker gene promoter. In one embodiment, the activity of an epithelial biomarker gene promoter may be assessed by inclusion of a human epithelial biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells such that said promoter reporter activity can be monitored by reporter gene expression level or activity. In one embodiment, the epithelial biomarker gene promoter-reporter gene construct is a human E-cadherin promoter-firefly luciferase construct. Additional examples of epithelial promoters that may be used include the promoters of the following genes: human ELF3 (i.e. E74-like factor 3 (ets domain transcription factor, epithelial-specific), GeneID: 1999). In an alternative embodiment, RNA transcript splicing mechanisms that are unique to human epithelial cells (Savagner, P. et al. (1994) Mol Biol Cell. 5(8):851-862; Oltean, S. et al. (2006) Proc Natl Acad Sci USA. 103(38):14116-14121; Ghigna, C. et al. (2005) Mol. Cell. 20(6):881-890; Bonano, V. I. et al. (2007) Nat. Protoc. 2(9):2166-2181), and do not operate after EMT, can be utilized as markers of EMT status. For example, by including the sequences necessary for such epithelial-specific splicing into a promoter-luciferase construct incorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferase construct) such that active luciferase will only be expressed in the epithelial state, induction of EMT can readily be monitored by a decrease in luciferase expression or activity. In an alternative embodiment, miRNAs that are expressed specifically in human epithelial cells (e.g. see Hurteau, G. J. et al. (2007) Cancer Research 67:7972-7976; Christoffersen, N. R. et al. (2007) RNA 13:1172-1178; Shell, S. et al (2007) Proc Natl. Acad. Sci. 104(27):11400-11405), that degrade or diminish translation of transcripts containing complementary nucleic acid sequences, can be utilized as markers of EMT status. For example, by including sequences complementary to such miRNAs into a promoter-luciferase construct incorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferase construct) such that active luciferase will not be expressed in the epithelial state, induction of EMT can readily be monitored by an increase in luciferase expression or activity. Similarly, in any of the other methods described herein involving a protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, the biomarker whose level is indicative of the EMT status of the sample tumor cells can be any of the examples described above.

In the above methods the biomarker whose level is indicative of the EMT status of the sample tumor cells is for example a mesenchymal cell biomarker, e.g. vimentin, fibronectin, N-cadherin, zeb1, twist, FOXC2 or snail, or the activity of a mesenchymal biomarker gene promoter. In one embodiment, the activity of a mesenchymal biomarker gene promoter may be assessed by inclusion of a human mesenchymal biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells such that said promoter reporter activity can be monitored by reporter gene expression level or activity. In one embodiment, the mesenchymal biomarker gene promoter-reporter gene construct is a human vimentin promoter-firefly luciferase construct. Additional examples of mesenchymal promoters that may be used include the promoters from the following human genes: S100A4 (i.e. S100 calcium binding protein A4 (a.k.a. FSP1), GeneID: 6275), SPARC (i.e. secreted protein, acidic, cysteine-rich (osteonectin), GeneID: 6678), IL-11 (i.e. interleukin 11, GeneID: 3589), PCOLCE2 (i.e. procollagen C-endopeptidase enhancer 2, GeneID: 26577), COL6A2 (i.e. collagen, type VI, alpha 2, GeneID: 1292), TFPI2 (i.e. tissue factor pathway inhibitor 2), GeneID: 7980), FBN1 (i.e. fibrillin 1, GeneID: 2200), Zeb1 (i.e. zinc finger E-box binding homeobox 1, GeneID: 6935), and CHST2 (i.e. carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2, GeneID: 9435). In an alternative embodiment, RNA transcript splicing mechanisms that are unique to human mesenchymal cells (Savagner, P. et al. (1994) Mol Biol Cell. 5(8):851-862; Oltean, S. et al. (2006) Proc Natl Acad Sci USA. 103(38):14116-14121; Ghigna, C. et al. (2005) Mol. Cell. 20(6):881-890; Bonano, V. I. et al. (2007) Nat. Protoc. 2(9):2166-2181), and do not operate prior to EMT, in epithelial cells, can be utilized as markers of EMT status. For example, by including the sequences necessary for such mesenchymal-specific splicing into a promoter-luciferase construct incorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferase construct) such that active luciferase will only be expressed in the mesenchymal state, induction of EMT can readily be monitored by an increase in luciferase expression or activity. In an alternative embodiment, miRNAs that are expressed specifically in human mesenchymal cells (e.g. see Hurteau, G. J. et al. (2007) Cancer Research 67:7972-7976; Christoffersen, N. R. et al. (2007) RNA 13:1172-1178; Shell, S. et al (2007) Proc Natl. Acad. Sci. 104(27):11400-11405), that degrade or diminish translation of transcripts containing complementary nucleic acid sequences, can be utilized as markers of EMT status. For example, by including sequences complementary to such miRNAs into a promoter-luciferase construct incorporated into CFPAC-1 cells (e.g. a CMV promoter-firefly luciferase construct) such that active luciferase will not be expressed in the mesenchymal state, induction of EMT can readily be monitored by a decrease in luciferase expression or activity. Similarly, in any of the other methods described herein involving a protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, the biomarker whose level is indicative of the EMT status of the sample tumor cells can be any of the examples described above.

In any of the methods or cell preparations described herein involving a biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells for monitoring biomarker promoter activity, more than one biomarker gene promoter-reporter gene construct may be employed so that multiple biomarkers may be simultaneously monitored in order to assess EMT status. For example, in one embodiment an epithelial biomarker gene promoter-reporter gene construct and a mesenchymal biomarker gene promoter-reporter gene construct are both used such that decreases in epithelial biomarker gene promoter activity and increases in mesenchymal biomarker gene promoter activity can both be monitored during EMT. For example, the epithelial biomarker gene promoter-reporter gene construct may be an E-cadherin promoter-firefly luciferase construct and the mesenchymal biomarker gene promoter-reporter gene construct may be a vimentin promoter-renilla luciferase construct. By using two different reporter genes that can be independently monitored (e.g. two luciferases that produce products that take part in luminescent reactions involving the emission of light of different characteristic wavelengths; e.g. see Hawkins, E. H. et al. (2002) Dual-Glo™ Luciferase Assay System: Convenient dual-reporter measurements in 96- and 384-well plates. Promega Notes 81, 22-6; Nieuwenhuijsen B W. et al. (2004) J Biomol Screen. 8, 676-84), both promoters can be monitored simultaneously. Similarly, two or more of the biomarkers described herein above involving epithelial or mesenchymal specific miRNAs or splicing mechanisms can be simultaneously monitored by using two different reporter genes that can be independently monitored.

In any of the methods or cell preparations described herein involving a biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells for monitoring biomarker promoter activity by assessing reporter gene expression level, the reporter gene can be any heterologous gene that expresses a protein whose level is readily determined by measuring expressed protein or enzymic activity. Suitable reporter genes include firefly (Photinus pyralis) luciferase, Renilla (Renilla reniformis) luciferase, Gaussia (Gaussia princeps) luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), etc. (e.g. see Hawkins, E. H. et al. (2002) Dual-Glo™ Luciferase Assay System: Convenient dual-reporter measurements in 96- and 384-well plates. Promega Notes 81, 22-6; Nieuwenhuijsen B W. et al. (2004) J. Biomol. Screen. 8, 676-84; Verhaegen M. and Christopoulos T. K. (2002) Anal. Chem., 74:4378-4385; Tannous, B. A., et al. (2005) Mol. Ther., 11:435-443; Hoffmann, R. M. (2004) Acta Histochemica 106(2):77-87); Hoffmann, R. M. (2008) Methods in Cell Biol. 85:485-495). Gaussia luciferase is a protein that is secreted from cells where it is expressed, hence it potentially allows, in any of the methods of the invention, the monitoring of reporter activity secreted into in the growth medium of cells in culture, or into the blood, or other biological fluids, from cells growing in vivo (e.g. tumor xenografts).

In one embodiment of the above methods, the sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, is an in vivo sample, such as, for example, a xenograft growing in an animal (e.g. immunocompromised mice or rats). Similarly, in any of the other methods or cell preparations described herein involving a protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, the sample of cells of the epithelial tumor cell line CFPAC-1 can be an in vivo sample, as described above.

The present invention also provides a method of identifying an agent that inhibits tumor cells that have undergone an epithelial to mesenchymal transition, comprising: contacting a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a compound that induces the expression of said protein such that an epithelial-to-mesenchymal transition is induced in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition. An alternative embodiment of this method comprises, after the step of determining whether the test agent inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, the additional steps of determining whether an agent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, also inhibits epithelial CFPAC-1 tumor cell growth, and thus determining whether it is an agent that specifically inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition. In an embodiment of the above methods, an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition is determined to do so by stimulating apoptosis of said tumor cells. In another embodiment of the above methods, an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition is determined to do so by inhibiting proliferation of said tumor cells.

The present invention also provides a method of identifying an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a compound that induces the expression of said protein such that an epithelial-to-mesenchymal transition is induced in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition.

The present invention also provides a tumor cell preparation for use in the identification of anti-cancer agents, wherein said tumor cell preparation comprises: a sample of cells of the epithelial tumor cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells. In one embodiment, the tumor cell preparation also comprises a mesenchymal biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells so that the activity of the mesenchymal biomarker gene promoter can be assessed by monitoring reporter gene level or activity. In one embodiment, the mesenchymal biomarker gene promoter-reporter gene construct is a vimentin promoter-firefly luciferase construct. In another embodiment, the tumor cell preparation comprises a epithelial biomarker gene promoter-reporter gene construct in the engineered CFPAC-1 cells so that the activity of the epithelial biomarker gene promoter can be assessed by monitoring reporter gene level or activity. In one embodiment, the epithelial biomarker gene promoter-reporter gene construct is an E-cadherin promoter-firefly luciferase construct. In the above cell preparations the protein that is inducibly expressed and stimulates an epithelial to mesenchymal transition in CFPAC-1 cells can be any of those identified for use in the methods described herein above that utilize such a cell preparation (e.g. Snail, Zeb1 etc). In one embodiment of this cell preparation, a Tet-regulated promoter is used to inducibly express (e.g. using doxycyclin, or tetracycline) the protein that stimulates an epithelial to mesenchymal transition in the CFPAC-1 cells. In the above embodiments, the biomarker gene promoter-reporter gene construct is stably expressed by the engineered CFPAC-1 cell. Accordingly, this invention provides, for use in the identification of anti-cancer agents, both an epithelial CFPAC-1 tumor cell preparation, prior to induction of a protein that stimulates an epithelial to mesenchymal transition, and a mesenchymal-like tumor cell preparation, after induction of a protein that stimulates an epithelial to mesenchymal transition. As described herein, these cell preparations can be used for screening test agents to identify agents that inhibit either cell type, or to find agents that will inhibit EMT or stimulate MET.

In the context of the methods of this invention, epithelial or mesenchymal biomarkers expressed by a tumor cell can include molecular and cellular markers that indicate the transition state of the tumor cell. In a preferred embodiment the biomarker is an individual marker protein, or its encoding mRNA, characteristic of the particular transition state of the tumor cell, i.e. a tumor cell exhibiting epithelial or mesenchymal characteristics. In an alternative embodiment, in certain circumstances the biomarker may be a characteristic morphological pattern produced in the tumor cell by cellular macromolecules that is characteristic of either an epithelial or mesenchymal condition. Thus, morphometric cell analysis can be used to provide information on epithelial or mesenchymal status of tumor cells. In an additional embodiment the biomarker that indicates the transition state of the tumor cell is methylation of the E-Cadherin gene (CDH1) promoter. CDH1 promoter methylation indicates that tumor cells have undergone an EMT transition.

TABLE 1 Molecular Biomarker Gene Identification Human Biomarker NCBI GeneID¹ NCBI RefSeq² E-cadherin 999 NP_004351 Brk 5753 NP_005966 γ-catenin 3728 NP_002221 α1-catenin 1495 NP_001894 α2-catenin 1496 NP_004380 α3-catenin 29119 NP_037398 keratin 8 3856 NP_002264 keratin 18 3875 NP_000215 vimentin 7431 NP_003371 fibronectin 1 2335 NP_002017 fibrillin-1 2200 NP_000129 fibrillin-2 2201 NP_001990 collagen alpha2(IV) 1284 NP_001837 collagen alpha2(V) 1290 NP_000384 LOXL1 4016 NP_005567 nidogen 4811 NP_002499 C11orf9 745 NP_037411 tenascin 3371 NP_002151 N-cadherin 1000 NP_001783 ¹The NCBI GeneID number is a unique identifier of the biomarker gene from the NCBI Entrez Gene database record (National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, 8600 Rockville Pike, Building 38A, Bethesda, MD 20894; Internet address http://www.ncbi.nlm.nih.gov/). ²The NCBI RefSeq (Reference Sequence) is an example of a sequence expressed by the biomarker gene.

TABLE 2 Molecular Biomarker Gene Identification Human Biomarker³ NCBI GeneID¹ NCBI RefSeq² Epithelial keratin K1 3848 NP_006112 keratin K2 3849 NP_000414 keratin K3 3850 NP_476429 keratin K4 3851 NP_002263 keratin K5 3852 NP_000415 keratin K6a 3853 NP_005545 keratin K6b 3854 NP_005546 keratin K6c 286887 NP_775109 keratin K7 3855 NP_005547 keratin K8 3856 NP_002264 keratin K9 3857 NP_000217 keratin K10 3858 NP_000412 keratin K12 3859 NP_000214 keratin K13 3860 NP_002265 keratin K14 3861 NP_000517 keratin K15 3866 NP_002266 keratin K16 3868 NP_005548 keratin K17 3872 NP_000413 keratin K18 3875 NP_000215 keratin K19 3880 NP_002267 keratin K20 54474 NP_061883 keratin K23 25984 NP_056330 keratin K24 192666 NP_061889 keratin K25 147183 NP_853512 keratin K26 353288 NP_853517 keratin K27 342574 NP_853515 keratin K28 162605 NP_853513 keratin K71 112802 NP_258259 keratin K72 140807 NP_542785 keratin K73 319101 NP_778238 keratin K74 121391 NP_778223 keratin K75 9119 NP_004684 keratin K76 51350 NP_056932 keratin K77 374454 NP_778253 keratin K78 196374 NP_775487 keratin K79 338785 NP_787028 keratin K80 144501 NP_001074961 Mesenchymal vimentin 7431 NP_003371 ¹The NCBI GeneID number is a unique identifier of the biomarker gene from the NCBI Entrez Gene database record (National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, 8600 Rockville Pike, Building 38A, Bethesda, MD 20894; Internet address http://www.ncbi.nlm.nih.gov/). ²The NCBI RefSeq (Reference Sequence) is an example of a sequence expressed by the biomarker gene. ³The new consensus nomenclature has been used herein when referring to keratins (see Schweizer, J. et al. (2006) J. Cell Biol. 174(2): 169-174). Former names for these proteins can be found in the latter reference, and at the Human Intermediate Filament Database (http://www.interfil.org/index.php). N.B. Epithelial keratins or cytokeratins are intermediate filament keratins. The terms “keratin” and “cytokeratin” are used synonymously herein. When refering to specific keratins in the text herein the “K” in the standard keratin designation (as in Table 2) is generally dropped (e.g. keratin K8 = keratin 8).

Table 1 lists genes coding for examples of epithelial or mesenchymal molecular biomarkers that can be used in the practice of the methods of the invention described herein. The epithelial or mesenchymal molecular biomarkers can include any product expressed by these genes, including variants thereof, e.g. expressed mRNA or protein, splice variants, co- and post-translationally modified proteins, polymorphic variants etc. In one embodiment the biomarker is the embryonal EDB⁺ fibronectin, a splice variant expressed by the fibronectin 1 gene (Kilian, O. et al. (2004) Bone 35(6):1334-1345). A possible advantage of determining this fetal form of fibronectin is that one could readily distinguish mesenchymal-like tumors from surrounding stromal tissue. Table 2 lists genes coding for examples of molecular biomarkers that can be used in the practice of certain embodiments of the methods of the invention described herein wherein co-expression of epithelial keratin(s) and the mesenchymal biomarker vimentin at similar levels is indicative of a mesenchymal-like cell. The molecular biomarkers can include any product expressed by these genes, including variants thereof, e.g. expressed mRNA or protein, splice variants, co- and post-translationally modified proteins, polymorphic variants etc.

In another embodiment of any of the methods of the invention described herein, the mesenchymal biomarker utilized in the method is selected from the human transcriptional repressors Snail (NCBI GeneID 6615), Zeb1 (NCBI GeneID 6935), Twist (NCBI GeneID 7291), Sip 1 NCBI GeneID 8487), and Slug (NCBI GeneID 6591).

In another embodiment of the methods of this invention the mesenchymal biomarker is methylation of the promoter of a gene whose transcription is repressed as a result of EMT in the tumor cell. In the context of this method high levels of a tumor cell mesenchymal biomarker essentially means readily detectable methylation of the promoter (e.g. a strong signal during detection of a methylation-specific PCR-amplified nucleic acid product derived from a promoter methylation site), whereas low levels of a tumor cell mesenchymal biomarker essentially means no detectable or low methylation of the promoter (e.g. no, or a comparatively weak, signal during detection of a methylation-specific PCR-amplified nucleic acid product derived from a promoter methylation site). In one embodiment of this method the gene whose transcription is repressed as a result of EMT in the tumor cell is the E-Cadherin gene (i.e. CDH1; NCBI GeneID 999). In another embodiment of this method the gene whose transcription is repressed as a result of EMT in the tumor cell is the γ-catenin gene (i.e. NCBI GeneID 3728). In another embodiment of this method the gene whose transcription is repressed as a result of EMT in the tumor cell is an α-catenin gene (e.g. NCBI GeneID 1495, 1496, or 29119). In another embodiment of this method the gene whose transcription is repressed as a result of EMT in the tumor cell is a cytokeratin gene (e.g. NCBI GeneID 3856 (keratin 8) or 3875 (keratin 18)).

Examples of additional epithelial markers that can be used in any of the methods of this invention include phospho-14-3-3 epsilon, 14-3-3 gamma (KCIP-1), 14-3-3 sigma (Stratifin), 14-3-3 zeta/delta, phospho-serine/threonine phosphatase 2A, 4F2hc(CD98 antigen), adenine nucleotide translocator 2, annexin A3, ATP synthase beta chain, phospho-insulin receptor substrate p53/p54, Basigin (CD147 antigen), phospho-CRK-associated substrate (p130Cas), Bcl-X, phospho-P-cadherin, phospho-calmodulin (CaM), Calpain-2 catalytic subunit, Cathepsin D, Cofilin-1, Calpain small subunit 1, Catenin beta-1, Catenin delta-1 (p 120 catenin), Cystatin B, phospho-DAZ-associated protein 1, Carbonyl reductase [NADPH], Diaphanous-related formin 1 (DRF1), Desmoglein-2, Elongation factor 1-delta, phospho-p185erbB2, Ezrin (p81), phospho-focal adhesion kinase 1, phospho-p94-FER (c-FER)., Filamin B, phospho-GRB2-associated binding protein 1, Rho-GDI alpha, phospho-GRB2, GRP 78, Glutathione S-transferase P, 3-hydroxyacyl-CoA dehydrogenase, HSP 90-alpha, HSP70.1, eIF3 p110, eIF-4E, Leukocyte elastase inhibitor, Importin-4, Integrin alpha-6, Integrin beta-4, phospho-Cytokeratin 17, Cytokeratin 19, Cytokeratin 7, Casein kinase I, alpha, Protein kinase C, delta, Pyruvate kinase, isozymes M1/M2, phospho-Erbin, LIM and SH3 domain protein 1 (LASP-1), 4F21c (CD98 light chain), L-lactate dehydrogenase A chain, Galectin-3, Galectin-3 binding protein, phospho-LIN-7 homolog C, MAP (APC-binding protein EB1), Maspin precursor (Protease inhibitor 5), phospho-Met tyrosine kinase (HGF receptor), Mixed-lineage leukemia protein 2, Monocarboxylate transporter 4, phospho-C-Myc binding protein (AMY-1), Myosin-9, Myosin light polypeptide 6, Nicotinamide phosphoribosyltransferase, Niban-like protein (Meg-3), Ornithine aminotransferase, phospho-Occludin, Ubiquitin thiolesterase, PAF acetylhydrolase IB beta subunit, phospho-partitioning-defective 3 (PAR-3), phospho-programmed cell death 6-interacting protein, phospho-Programmed cell death protein 6, Protein disulfide-isomerase, phospho-plakophilin-2, phospho-plakophilin-3, Protein phosphatase 1, Peroxiredoxin 5, Proteasome activator complex subunit 1, Prothymosin alpha, Retinoic acid-induced protein 3, phospho-DNA repair protein REV 1, Ribonuclease inhibitor, RuvB-like 1, S-100P, S-100L, Calcyclin, S100C, phospho-Sec23A, phospho-Sec23B, Lysosome membrane protein II (LIMP II), p60-Src, phospho-Amplaxin (EMS1), SLP-2, Gamma-synuclein, Tumor calcium signal transducer 1, Tumor calcium signal transducer 2, Transgelin-2, Transaldolase, Tubulin beta-2 chain, Translationally controlled (TCTP), Tissue transglutaminase, Transmembrane protein Tmp21, Ubiquitin-conjugating enzyme E2 N, UDP-glucosyltransferase 1, phospho-p6′-Yes, phospho-Tight junction protein ZO-1, AHNAK (Desmoyokin), phospho-ATP synthase beta chain, phospho-ATP synthase delta, Cold shock domain protein E1, Desmoplakin III, Plectin 1, phospho-Nectin 2 (CD112 antigen), phospho-p185-Ron, phospho-SHC1, E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, ST14, and other epithelial biomarkers known in the art (see for example, US Patent Application Publication 2007/0212738; U.S. Patent Application 60/923,463; U.S. Patent Application 60/997,514). Where the epithelial biomarker is a phospho-“protein” the extent of phosphorylation of the protein rather than the level of the protein per se is the parameter that is altered after EMT. The altered level of phosphorylation of these proteins is also understood to be due to changes in the level of phosphorylation of one or more tyrosine residues of the protein (US Patent Application Publication 2007/0212738).

Examples of additional mesenchymal markers that can be used in any of the methods of this invention include MMP9 (matrix-metalloproteinase 9; NCBI Gene ID No. 4318), MHC class I antigen A*1, Acyl-CoA desaturase, LANP-like protein (LANP-L), Annexin A6, ATP synthase gamma chain, BAG-family molecular chaperone regulator-2, phospho-Bullous pemphigoid antigen, phospho-Protein Clorf77, CDK1 (cdc2), phospho-Clathrin heavy chain 1, Condensin complex subunit 1,3,2-trans-enoyl-CoA isomerase, DEAH-box protein 9, phospho-Enhancer of rudimentary homolog, phospho-Fibrillarin, GAPDH muscle, GAPDH liver, Synaptic glycoprotein SC2, phospho-Histone H1.0, phospho-Histone H1.2, phospho-Histone H1.3, phospho-Histone H1.4, phospho-Histone H1.5, phospho-Histone H1x, phospho-Histone H2AFX, phospho-H1stone H2A.o, phospho-H1stone H2A.q, phospho-Histone H2A.z, phospho-Histone H2B.j, phospho-Histone H2B.r, phospho-Histone H4, phospho-HMG-17-like 3, phospho-HMG-14, phospho-HMG-17, phospho-HMGI-C, phospho-HMG-I/HMG-Y, phospho-Thyroid receptor interacting protein 7 (TRIP7), phospho-hnRNP H3, hnRNP C1/C2, hnRNP F, phospho-hnRNP G, eIF-5A, NFAT 45 kDa, Importin beta-3, cAMP-dependent PK1a, Lamin B1, Lamin A/C, phospho-Laminin alpha-3 chain, L-lactate dehydrogenase B chain, Galectin-1, phospho-Fez1, Hyaluronan-binding protein 1, phospho-Microtubule-actin crosslinking factor 1, Melanoma-associated antigen 4, Matrin-3, Phosphate carrier protein, Myosin-10, phospho-N-acylneuraminate cytidylyltransferase, phospho-NHP2-like protein 1, H/ACA ribonucleoprotein subunit 1, Nucleolar phosphoprotein p130, phospho-RNA-binding protein Nova-2, Nucleophosmin (NPM), NADH-ubiquinone oxidoreductase 39 kDa subunit, phospho-Polyadenylate-binding protein 2, Prohibitin, Prohibitin-2, Splicing factor Prp8, Polypyrimidine tract-binding protein 1, Parathymosin, Rab-2A, phospho-RNA-binding protein Raly, Putative RNA-binding protein 3, phospho-60S ribosomal protein L23, hnRNP A0, hnRNP A2/B1, hnRNP A/B, U2 small nuclear ribonucleoprotein B, phospho-Ryanodine receptor 3, phospho-Splicing factor 3A subunit 2, snRNP core protein D3, Nesprin-1, Tyrosine—tRNA ligase, phospho-Tankyrase 1-BP, Tubulin beta-3, Acetyl-CoA acetyltransferase, phospho-bZIP enhancing factor BEF (Aly/REF; Tho4), Ubiquitin, Ubiquitin carboxyl-terminal hydrolase 5, Ubiquinol-cytochrome c reductase, Vacuolar protein sorting 16, phospho-Zinc finger protein 64, phospho-AHNAK (Desmoyokin), ATP synthase beta chain, ATP synthase delta chain, phospho-Cold shock domain protein E1, phospho-Plectin 1, Nectin 2 (CD112 antigen), p185-Ron, SHC1, vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, embryonal EDB⁺ fibronectin, tubulin alpha-3, epimorphin, and other mesenchymal biomarkers known in the art, (see for example, US Patent Application Publication 2007/0212738; U.S. Patent Application 60/923,463; U.S. Patent Application 60/997,514). Where the mesenchymal biomarker is a phospho-“protein” the extent of phosphorylation of the protein rather than the level of the protein per se is the parameter that is altered after EMT. The altered level of phosphorylation of these proteins is also understood to be due to changes in the level of phosphorylation of one or more tyrosine residues of the protein (US Patent Application Publication 2007/0212738).

The biomarkers in the above lists of epithelial and mesenchymal biomarkers have been identified as being altered in expression level (or phosphoylation level for phospho-“proteins”) after EMT (see for example, US Patent Application Publication 2007/0212738, the contents of which are incorporated herein by reference; US Published Application 2006/0211060 (filed Mar. 16, 2006); Thomson, S. et al. (2005) Cancer Res. 65(20) 9455-9462; and Yauch, R. L. et al. (2005) Clin. Can. Res. 11(24) 8686-8698).

In the methods of this invention, biomarker expression level can be assessed relative to a control molecule whose expression level remains constant throughout EMT or when comparing tumor cells expressing either epithelial or mesenchymal transition states as indicated by molecular biomarkers (e.g. a “housekeeping” gene, such as GAPDH, β-actin, tubulin, or the like). Biomarker expression level can also be assessed relative to the other type of tumor cell biomarker (i.e. epithelial compared to mesenchymal), or to the biomarker level in non-tumor cells of the same tissue, or another cell or tissue source used as an assay reference.

In the methods of this invention, the level of an epithelial or mesenchymal biomarker expressed by a tumor cell can be assessed by using any of the standard bioassay procedures known in the art for determination of the level of expression of a gene, including for example ELISA, RIA, immunopreciptation, immunoblotting, immunofluorescence microscopy, immunohistochemistry (IHC), RT-PCR, in situ hybridization, cDNA microarray, or the like, as described in more detail below. In an embodiment of any of these methods, their use is coupled with a method to isolate a particular cell population, e.g. laser capture microdissection (LCM). In an additional embodiment, FACS analysis can be used with immunofluorescence biomarker (e.g. E-cadherin) labeling to isolate and quantify cell populations expressing different epithelial or mesenchymal biomarkers, and thus for example the percentage of cells that have undergone an EMT can be estimated (e.g. see Xu, Z. et al. (2003) Cell Research 13(5):343-350).

In the methods of this invention, the expression level of a tumor cell epithelial or mesenchymal biomarker in vivo is preferably assessed by assaying a tumor biopsy. In one embodiment the biopsy comprises samples taken from multiple areas of the tumor, or a method (e.g. core needle biopsy) that samples different areas of the tumor, thus ensuring that when the tumor is of a heterogeneous nature with respect to the types of cells it contains, that a representative biopsy is obtained. In an alternative embodiment, given that a tumor may be heterogeneous with respect to the EMT status of the cells it contains, the methods of this invention are preferably applied separately to different cell types (e.g. using IHC, or an analysis method coupled with a step to isolate a particular cell population). Alternatively, by employing cell surface epithelial and/or mesenchymal biomarker antibodies (e.g. to E-cadherin), FACS analysis can be used to isolate and quantify the numbers of tumor cells at different stages of EMT.

However, in an alternative embodiment, expression level of the tumor cell biomarker can be assessed in bodily fluids or excretions containing detectable levels of biomarkers originating from the tumor or tumor cells. Bodily fluids or excretions useful in the present invention include blood, urine, saliva, stool, pleural fluid, lymphatic fluid, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. By blood it is meant to include whole blood, plasma, serum or any derivative of blood. Assessment of tumor epithelial or mesenchymal biomarkers in such bodily fluids or excretions can sometimes be preferred in circumstances where an invasive sampling method is inappropriate or inconvenient.

For assessment of tumor cell epithelial or mesenchymal biomarker expression, tumor samples containing tumor cells, or proteins or nucleic acids produced by these tumor cells, may be used in the methods of the present invention. In these embodiments, the level of expression of the biomarker can be assessed by assessing the amount (e.g. absolute amount or concentration) of the marker in a tumor cell sample, e.g., a tumor biopsy obtained from an animal, or another sample containing material derived from the tumor (e.g. blood, serum, urine, or other bodily fluids or excretions as described herein above). The cell sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the marker in the sample. Likewise, tumor biopsies may also be subjected to post-collection preparative and storage techniques, e.g., fixation.

Determination of epithelial or mesenchymal biomarker levels in in vivo studies can be assessed by a number of different approaches, including direct analysis of proteins that segregate as epithelial related (e.g. E-cadherin) or mesenchymal related (e.g. vimentin, Zeb1) biomarkers. An advantage of this approach is that EMT markers are read directly, and the relative amounts of cell populations expressing epithelial or mesenchymal biomarkers can readily be examined and quantified, by for example FACS analysis (e.g. see Xu, Z. et al. (2003) Cell Research 13(5):343-350). However, this approach also requires sufficient quantities of cells or tissue in order to perform an analysis (e.g. immunohistochemistry). Sufficient quantities of tissue may be difficult to obtain from certain procedures such as FNA (fine needle aspiration). Core biopsies provide larger amounts of tissue, but are sometimes not readily available. Alternatively, these EMT biomarkers could be evaluated based upon the expression level of their encoding RNA transcripts using a quantitative PCR based approach. An advantage of this approach is that very few tumor cells are required for this measurement, and it is very likely that sufficient material may be obtained via an FNA. However, here the transcript levels for a given biomarker may be derived from both tumor cells as well as infiltrating stromal cells from the tumor. Given that stromal cells also express mesenchymal cell markers, this may obscure detection of the EMT status for tumor cells. Use of in situ hybridization (e.g. FISH) or tissue microdisection may be useful here to overcome this potential limitation.

Given that the expression level of E-cadherin is a hallmark of the EMT status for a tumor cell, EMT may also be evaluated based upon the methylation status of the E-cadherin promoter, as described herein. Methylation silences transcription, and so a high level of methylation correlates with a mesenchymal-like state. A potential benefit of this approach is that, like measurement of transcript levels, measuring the methylation status of DNA would likely require very little material. Sufficient material could likely be obtained from an FNA and would not require a core biopsy. Additionally, since this approach involves evaluation of DNA and not RNA, it is likely to be a more stable read-out over time, such as during medium or long term storage of a sample.

In the methods of the invention, one can detect expression of biomarker proteins having at least one portion which is displayed on the surface of tumor cells which express it. It is a simple matter for the skilled artisan to determine whether a marker protein, or a portion thereof, is exposed on the cell surface. For example, immunological methods may be used to detect such proteins on whole cells, or well known computer-based sequence analysis methods may be used to predict the presence of at least one extracellular domain (i.e. including both secreted proteins and proteins having at least one cell-surface domain). Expression of a marker protein having at least one portion which is displayed on the surface of a cell which expresses it may be detected without necessarily lysing the tumor cell (e.g. using a labeled antibody which binds specifically with a cell-surface domain of the protein).

Expression of a biomarkers described in this invention may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed nucleic acid or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

In one embodiment, expression of a biomarker is assessed using an antibody (e.g. a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g. an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g. biotin-streptavidin}), or an antibody fragment (e.g. a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a biomarker protein or fragment thereof, including a biomarker protein which has undergone either all or a portion of post-translational modifications to which it is normally subjected in the tumor cell (e.g. glycosylation, phosphorylation, methylation etc.).

In another embodiment, expression of a biomarker is assessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from tumor cells, and by hybridizing the mRNA/cDNA with a reference polynucleotide which is a complement of a biomarker nucleic acid, or a fragment thereof cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more biomarkers can likewise be detected using quantitative PCR to assess the level of expression of the biomarker(s). Alternatively, any of the many known methods of detecting mutations or variants (e.g. single nucleotide polymorphisms, deletions, etc.) of a biomarker of the invention may be used to detect occurrence of a biomarker in a tumor cell.

In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a biomarker nucleic acid. If polynucleotides complementary to or homologous with are differentially detectable on the substrate (e.g. detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of biomarkers can be assessed simultaneously using a single substrate (e.g. a “gene chip” microarray of polynucleotides fixed at selected positions). When a method of assessing biomarker expression is used which involves hybridization of one nucleic acid with another, it is preferred that the hybridization be performed under stringent hybridization conditions.

When a plurality of biomarkers of the invention are used in the methods of the invention, the level of expression of each biomarker in tumor cells induced to undergo EMT can be compared with the level of expression of each of the plurality of biomarkers in non-induced samples of the same type, either in a single reaction mixture (i.e. using reagents, such as different fluorescent probes, for each biomarker) or in individual reaction mixtures corresponding to one or more of the biomarkers.

An exemplary method for detecting the presence or absence of a biomarker protein or nucleic acid in a biological sample involves obtaining a biological sample (e.g. a tumor-associated body fluid) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a biomarker protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. In vivo techniques for detection of mRNA include polymerase chain reaction (PCR), Northern hybridizations and in situ hybridizations. Furthermore, in vivo techniques for detection of a biomarker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a biomarker, and a probe, under appropriate conditions and for a time sufficient to allow the biomarker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoring the biomarker or probe onto a solid phase support, also referred to as a substrate, and detecting target biomarker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of biomarker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to a solid phase. These include, without limitation, biomarker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the biomarker or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of biomarker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

In one embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect biomarker/probe complex formation without further manipulation or labeling of either component (biomarker or probe), for example by utilizing the technique of fluorescence energy transfer (i.e. FET, see for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe to recognize a biomarker can be accomplished without labeling either assay component (probe or biomarker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S, and Urbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with biomarker and probe as solutes in a liquid phase. In such an assay, the complexed biomarker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, biomarker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the biomarker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and Tweed, S. A. J. Chromatogr B Biomed Sci Appl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

In a particular embodiment, the level of biomarker mRNA can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a biomarker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the biomarker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the biomarkers of the present invention.

An alternative method for determining the level of mRNA biomarker in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the tumor cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the biomarker.

As an alternative to making determinations based on the absolute expression level of the biomarker, determinations may be based on the normalized expression level of the biomarker. Expression levels are normalized by correcting the absolute expression level of a biomarker by comparing its expression to the expression of a gene that is not a biomarker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a tumor cell sample, to another sample, e.g., a non-tumor sample, or between samples from different sources, or between samples before and after induction of EMT.

Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a biomarker (e.g. a mesenchymal biomarker), the level of expression of the biomarker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the biomarker. The expression level of the biomarker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that biomarker. This provides a relative expression level.

In another embodiment of the present invention, a biomarker protein is detected. A preferred agent for detecting biomarker protein of the invention is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment or derivative thereof (e.g., Fab or F(ab′).sub.2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

Proteins from tumor cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether tumor cells express a biomarker of the present invention.

In one format, antibodies, or antibody fragments or derivatives, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from tumor cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

For ELISA assays, specific binding pairs can be of the immune or non-immune type. Immune specific binding pairs are exemplified by antigen-antibody systems or hapten/anti-hapten systems. There can be mentioned fluorescein/anti-fluorescein, dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin, peptide/anti-peptide and the like. The antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, e.g., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic. Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies. Exemplary non-immune pairs are biotin-streptavidin, intrinsic factor-vitamin B₁₂, folic acid-folate binding protein and the like.

A variety of methods are available to covalently label antibodies with members of specific binding pairs. Methods are selected based upon the nature of the member of the specific binding pair, the type of linkage desired, and the tolerance of the antibody to various conjugation chemistries. Biotin can be covalently coupled to antibodies by utilizing commercially available active derivatives. Some of these are biotin-N-hydroxy-succinimide which binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled to protein amine groups using 2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other standard methods of conjugation can be employed to couple monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC).

Homobifunctional crosslinkers, including the bifunctional imidoesters and bifunctional N-hydroxysuccinimide esters, are commercially available and are employed for coupling amine groups on one substance to amine groups on another. Heterobifunctional crosslinkers are reagents which possess different functional groups. The most common commercially available heterobifunctional crosslinkers have an amine reactive N-hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group. The most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens. One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.

The detectably-labeled antibody or detectably-labeled member of the specific binding pair is prepared by coupling to a reporter, which can be a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electrochemical materials. Two commonly used radioactive isotopes are ¹²⁵I and ³H. Standard radioactive isotopic labeling procedures include the chloramine T, lactoperoxidase and Bolton-Hunter methods for ¹²⁵I and reductive methylation for ³H. The term “detectably-labeled” refers to a molecule labeled in such a way that it can be readily detected by the intrinsic enzymic activity of the label or by the binding to the label of another component, which can itself be readily detected.

Enzymes suitable for use in this invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, luciferases, including firefly and renilla, β-lactamase, urease, green fluorescent protein (GFP) and lysozyme. Enzyme labeling is facilitated by using dialdehyde, carbodiimide coupling, homobifunctional crosslinkers and heterobifunctional crosslinkers as described above for coupling an antibody with a member of a specific binding pair.

The labeling method chosen depends on the functional groups available on the enzyme and the material to be labeled, and the tolerance of both to the conjugation conditions. The labeling method used in the present invention can be one of, but not limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avrameas and Ternynck, Immunochemistry 8, 1175 (1975), Ishikawa et al., J. Immunoassay 4(3):209-327 (1983) and Jablonski, Anal. Biochem. 148:199 (1985).

Labeling can be accomplished by indirect methods such as using spacers or other members of specific binding pairs. An example of this is the detection of a biotinylated antibody with unlabeled streptavidin and biotinylated enzyme, with streptavidin and biotinylated enzyme being added either sequentially or simultaneously. Thus, according to the present invention, the antibody used to detect can be detectably-labeled directly with a reporter or indirectly with a first member of a specific binding pair. When the antibody is coupled to a first member of a specific binding pair, then detection is effected by reacting the antibody-first member of a specific binding complex with the second member of the binding pair that is labeled or unlabeled as mentioned above.

Moreover, the unlabeled detector antibody can be detected by reacting the unlabeled antibody with a labeled antibody specific for the unlabeled antibody. In this instance “detectably-labeled” as used above is taken to mean containing an epitope by which an antibody specific for the unlabeled antibody can bind. Such an anti-antibody can be labeled directly or indirectly using any of the approaches discussed above. For example, the anti-antibody can be coupled to biotin which is detected by reacting with the streptavidin-horseradish peroxidase system discussed above.

In one embodiment of this invention biotin is utilized. The biotinylated antibody is in turn reacted with streptavidin-horseradish peroxidase complex. Orthophenylenediamine, 4-chloro-naphthol, tetramethylbenzidine (TMB), ABTS, BTS or ASA can be used to effect chromogenic detection.

In one immunoassay format for practicing this invention, a forward sandwich assay is used in which the capture reagent has been immobilized, using conventional techniques, on the surface of a support. Suitable supports used in assays include synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, e.g. aminated or carboxylated polystyrene, polyacrylamides, polyamides, polyvinylchloride, glass beads, agarose, or nitrocellulose.

Agents, or compositions comprising such agents, that inhibit tumor cells from undergoing an epithelial to mesenchymal transition, inhibit tumor cells that have undergone an epithelial to mesenchymal transition, or stimulate mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, identified by the methods described herein, can be used in methods for treating tumors or tumor metastases in a patient.

As used herein, the term “patient” preferably refers to a human in need of treatment with an anti-cancer agent for any purpose, and more preferably a human in need of such a treatment to treat cancer, or a precancerous condition or lesion. However, the term “patient” can also refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others, that are in need of treatment with an anti-cancer agent.

Anti-cancer agents identified by any of the methods described herein (or compositions comprising them) can be used to treat any of the following tumors or cancers: NSCL, breast, colon, or pancreatic cancer, lung cancer, bronchioloalveolar cell lung cancer, bone cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the ureter, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, chronic or acute leukemia, lymphocytic lymphomas, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

The term “refractory” as used herein is used to define a cancer for which treatment (e.g. chemotherapy drugs, biological agents, and/or radiation therapy) has proven to be ineffective. A refractory cancer tumor may shrink, but not to the point where the treatment is determined to be effective. Typically however, the tumor stays the same size as it was before treatment (stable disease), or it grows (progressive disease).

Anti-cancer agents identified by any of the methods described herein (or compositions comprising them) can be used to treat abnormal cell growth.

It will be appreciated by one of skill in the medical arts that the exact manner of administering to said patient of a therapeutically effective amount of the identified agent, e.g. in a combination of an EGFR kinase inhibitor and said agent, will be at the discretion of the attending physician. The mode of administration, including dosage, combination with other anti-cancer agents, timing and frequency of administration, and the like, may be affected by e.g. a diagnosis of a patient's likely responsiveness to an EGFR or IGFR kinase inhibitor, as well as the patient's condition and history. Thus, even patients diagnosed with tumors predicted to be relatively sensitive to e.g. an EGFR or IGFR kinase inhibitor as a single agent may still benefit from treatment with a combination of such a kinase inhibitor and the identified agent, optionally in combination with other anti-cancer agents, or other agents that may alter a tumor's sensitivity to kinase inhibitors.

In the context of this invention, an “effective amount” of an agent or therapy is as defined above. A “sub-therapeutic amount” of an agent or therapy is an amount less than the effective amount for that agent or therapy, but when combined with an effective or sub-therapeutic amount of another agent or therapy can produce a result desired by the physician, due to, for example, synergy in the resulting efficacious effects, or reduced side effects.

Additionally, the present invention provides a pharmaceutical composition comprising a combination of for example an EGFR or IGFR kinase inhibitor and the identified agent in a pharmaceutically acceptable carrier.

The invention also encompasses a pharmaceutical composition prepared by any of the methods described herein, that is comprised of an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, inhibit tumor cells that have undergone an epithelial to mesenchymal transition, or stimulate mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, identified by any of the methods described herein (i.e. “the identified agent”), in combination with a pharmaceutically acceptable carrier, and optionally in combination with one or more other anti-cancer agents (e.g. an EGFR, IGF-1R, RON or MET receptor tyrosine kinase inhibitor).

Methods of preparing pharmaceutical compositions are well known in the art, as for example described in references such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18^(th) edition (1990).

Preferably the composition is comprised of a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of the identified agent (including pharmaceutically acceptable salts of each component thereof).

Moreover, within this preferred embodiment, the invention encompasses a pharmaceutical composition for the treatment of disease, the use of which results in the inhibition of growth of neoplastic cells, benign or malignant tumors, or metastases, comprising a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of the identified agent (including pharmaceutically acceptable salts of each component thereof).

The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When a compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (cupric and cuprous), ferric, ferrous, lithium, magnesium, manganese (manganic and manganous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N′,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylameine, trimethylamine, tripropylamine, tromethamine and the like.

When a compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

The pharmaceutical compositions of the present invention comprise the identified agent (including pharmaceutically acceptable salts thereof) as active ingredient, a pharmaceutically acceptable carrier and optionally other therapeutic ingredients or adjuvants. Other therapeutic agents may include those cytotoxic, chemotherapeutic or anti-cancer agents, or agents which enhance the effects of such agents, as listed above. The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In practice, the compounds represented by the identified agent (including pharmaceutically acceptable salts thereof) of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the identified agent (including pharmaceutically acceptable salts thereof) may also be administered by controlled release means and/or delivery devices. The combination compositions may be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredients with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention may include a pharmaceutically acceptable carrier and a combination of the identified agent (including pharmaceutically acceptable salts thereof). The identified agent (including pharmaceutically acceptable salts thereof), can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. Other therapeutically active compounds may include those cytotoxic, chemotherapeutic or anti-cancer agents, or agents which enhance the effects of such agents, as listed above.

Thus in one embodiment of this invention, a pharmaceutical composition can comprise the identified agent in combination with an anticancer agent, wherein said anti-cancer agent is a member selected from the group consisting of alkylating drugs, antimetabolites, microtubule inhibitors, podophyllotoxins, antibiotics, nitrosoureas, hormone therapies, kinase inhibitors, activators of tumor cell apoptosis, and antiangiogenic agents.

The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenient pharmaceutical media may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or nonaqueous techniques.

A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient and each cachet or capsule preferably contains from about 0.05 mg to about 5 g of the active ingredient.

For example, a formulation intended for the oral administration to humans may contain from about 0.5 mg to about 5 g of active agent, compounded with an appropriate and convenient amount of carrier material that may vary from about 5 to about 95 percent of the total composition. Unit dosage forms will generally contain between from about 1 mg to about 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

Pharmaceutical compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a form suitable for topical sue such as, for example, an aerosol, cream, ointment, lotion, dusting powder, or the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations may be prepared, utilizing the identified agent (including pharmaceutically acceptable salts thereof) of this invention, via conventional processing methods. As an example, a cream or ointment is prepared by admixing hydrophilic material and water, together with about 5 wt % to about 10 wt % of the compound, to produce a cream or ointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing the identified agent (including pharmaceutically acceptable salts thereof) may also be prepared in powder or liquid concentrate form.

In the context of this invention, other anticancer agents includes, for example, other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effects of such agents, anti-hormonal agents, angiogenesis inhibitors, tumor cell pro-apoptotic or apoptosis-stimulating agents, signal transduction inhibitors, anti-proliferative agents, anti-HER2 antibody or an immunotherapeutically active fragment thereof, anti-proliferative agents, COX II (cyclooxygenase II) inhibitors, and agents capable of enhancing antitumor immune responses.

In the context of this invention, other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effects of such agents, include, for example: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g. CYTOXAN®), chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (C is P; e.g. PLATINOL®) busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites, such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®), 6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC), and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g. ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g. TAXOL®) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar, diverse antitumor agents. The following agents may also be used as additional agents: arnifostine (e.g. ETHYOL®), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g. DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicin lipo (e.g. DAUNOXOMEC), procarbazine, mitomycin, docetaxel (e.g. TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil.

As used herein, the term “anti-hormonal agent” includes natural or synthetic organic or peptidic compounds that act to regulate or inhibit hormone action on tumors. Antihormonal agents include, for example: steroid receptor antagonists, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, other aromatase inhibitors, 42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (e.g. FARESTON®); anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above; agonists and/or antagonists of glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasing hormone); the LHRH agonist goserelin acetate, commercially available as ZOLADEX® (AstraZeneca); the LHRH antagonist D-alaninamide N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N-6-(3-pyridinylcarbonyl)-L-lysyl-N-6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N-6-(1-methylethyl)-L-lysyl-L-proline (e.g. ANTIDE®, Ares-Serono); the LHRH antagonist ganirelix acetate; the steroidal anti-androgens cyproterone acetate (CPA) and megestrol acetate, commercially available as MEGACE® (Bristol-Myers Oncology); the nonsteroidal anti-androgen flutamide (2-methyl-N-[4,20-nitro-3-(trifluoromethyl) phenylpropanamide), commercially available as EULEXIN® (Schering Corp.); the non-steroidal anti-androgen nilutamide, (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione); and antagonists for other non-permissive receptors, such as antagonists for RAR, RXR, TR, VDR, and the like.

Anti-angiogenic agents include, for example: VEGFR inhibitors, such as SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA), or as described in, for example International Application Nos. WO 99/24440, WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437, and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504 and 6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland, Wash., USA); angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.); and antibodies to VEGF, such as bevacizumab (e.g. AVASTIN™, Genentech, South San Francisco, Calif.), a recombinant humanized antibody to VEGF; integrin receptor antagonists and integrin antagonists, such as to α_(v)β₃, α_(v)β₅ and α_(v)β₆ integrins, and subtypes thereof, e.g. cilengitide (EMD 121974), or the anti-integrin antibodies, such as for example α_(v)β₃ specific humanized antibodies (e.g. VITAXIN®); factors such as IFN-alpha (U.S. Pat. Nos. 41,530,901, 4,503,035, and 5,231,176); angiostatin and plasminogen fragments (e.g. kringle 1-4, kringle 5, kringle 1-3 (O'Reilly, M. S. et al. (1994) Cell 79:315-328; Cao et al. (1996) J. Biol. Chem. 271: 29461-29467; Cao et al. (1997) J. Biol. Chem. 272:22924-22928); endostatin (O'Reilly, M. S. et al. (1997) Cell 88:277; and International Patent Publication No. WO 97/15666); thrombospondin (TSP-1; Frazier, (1991) Curr. Opin. Cell Biol. 3:792); platelet factor 4 (PF4); plasminogen activator/urokinase inhibitors; urokinase receptor antagonists; heparinases; fumagillin analogs such as TNP-4701; suramin and suramin analogs; angiostatic steroids; bFGF antagonists; flk-1 and flt-1 antagonists; anti-angiogenesis agents such as MMP-2 (matrix-metalloproteinase 2) inhibitors and MMP-9 (matrix-metalloproteinase 9) inhibitors. Examples of useful matrix metalloproteinase inhibitors are described in International Patent Publication Nos. WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, and WO 99/07675, European Patent Publication Nos. 818,442, 780,386, 1,004,578, 606,046, and 931,788; Great Britain Patent Publication No. 9912961, and U.S. Pat. Nos. 5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred, are those that selectively inhibit MMP-2 and/or MMP-9 relative to the other matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Signal transduction inhibitors include, for example: erbB2 receptor inhibitors, such as organic molecules, or antibodies that bind to the erbB2 receptor, for example, trastuzumab (e.g. HERCEPTIN®); inhibitors of other protein tyrosine-kinases, e.g. imitinib (e.g. GLEEVEC®); ras inhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; cyclin dependent kinase inhibitors; protein kinase C inhibitors; and PDK-1 inhibitors (see Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313, for a description of several examples of such inhibitors, and their use in clinical trials for the treatment of cancer).

ErbB2 receptor inhibitors include, for example: ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome plc), monoclonal antibodies such as AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands, Tex., USA) and 2B-1 (Chiron), and erbB2 inhibitors such as those described in International Publication Nos. WO 98/02434, WO 99/35146, WO 99/35132, WO 98/02437, WO 97/13760, and WO 95/19970, and U.S. Pat. Nos. 5,587,458, 5,877,305, 6,465,449 and 6,541,481.

Antiproliferative agents include, for example: Inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFR, including the compounds disclosed and claimed in U.S. Pat. Nos. 6,080,769, 6,194,438, 6,258,824, 6,586,447, 6,071,935, 6,495,564, 6,150,377, 6,596,735 and 6,479,513, and International Patent Publication WO 01/40217. Antiproliferative agents also include inhibitors of the receptor tyrosine kinases IGF-1R and FGFR.

Examples of useful COX-II inhibitors include alecoxib (e.g. CELEBREX™), valdecoxib, and rofecoxib. Agents capable of enhancing antitumor immune responses include, for example: CTLA4 (cytotoxic lymphocyte antigen 4) antibodies (e.g. MDX-CTLA4), and other agents capable of blocking CTLA4. Specific CTLA4 antibodies that can be used in the present invention include those described in U.S. Pat. No. 6,682,736.

The present invention further provides a method for treating tumors or tumor metastases in a patient, comprising administering to the patient simultaneously or sequentially a therapeutically effective amount of the identified agent described herein above and optionally one or more other anticancer agents.

Dosage levels for the identified agents of this invention will be as described herein, but will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The use of the cytotoxic and other anticancer agents described above in chemotherapeutic regimens is generally well characterized in the cancer therapy arts, and their use herein falls under the same considerations for monitoring tolerance and effectiveness and for controlling administration routes and dosages, with some adjustments. For example, the actual dosages of the cytotoxic agents may vary depending upon the patient's cultured cell response determined by using histoculture methods. Generally, the dosage will be reduced compared to the amount used in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the ranges recommended by the manufacturer, and where indicated by in vitro responses or responses in animal models, can be reduced by up to about one order of magnitude concentration or amount. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based on the in vitro responsiveness of the primary cultured malignant cells or histocultured tissue sample, or the responses observed in the appropriate animal models.

As used herein, the term “EGFR kinase inhibitor” refers to any EGFR kinase inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the EGF receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to EGFR of its natural ligand. Such EGFR kinase inhibitors include any agent that can block EGFR activation or any of the downstream biological effects of EGFR activation that are relevant to treating cancer in a patient. Such an inhibitor can act by binding directly to the intracellular domain of the receptor and inhibiting its kinase activity. Alternatively, such an inhibitor can act by occupying the ligand binding site or a portion thereof of the EGF receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced. Alternatively, such an inhibitor can act by modulating the dimerization of EGFR polypeptides, or interaction of EGFR polypeptide with other proteins, or enhance ubiquitination and endocytotic degradation of EGFR. EGFR kinase inhibitors include but are not limited to low molecular weight inhibitors, antibodies or antibody fragments, peptide or RNA aptamers, antisense constructs, small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), and ribozymes. In a preferred embodiment, the EGFR kinase inhibitor is a small organic molecule or an antibody that binds specifically to the human EGFR.

EGFR kinase inhibitors include, for example quinazoline EGFR kinase inhibitors, pyrido-pyrimidine EGFR kinase inhibitors, pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo-pyrimidine EGFR kinase inhibitors, pyrazolo-pyrimidine EGFR kinase inhibitors, phenylamino-pyrimidine EGFR kinase inhibitors, oxindole EGFR kinase inhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFR kinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFR kinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as those described in the following patent publications, and all pharmaceutically acceptable salts and solvates of said EGFR kinase inhibitors: International Patent Publication Nos. WO 96/33980, WO 96/30347, WO 97/30034, WO 97/30044, WO 97/38994, WO 97/49688, WO 98/02434, WO 97/38983, WO 95/19774, WO 95/19970, WO 97/13771, WO 98/02437, WO 98/02438, WO 97/32881, WO 98/33798, WO 97/32880, WO 97/3288, WO 97/02266, WO 97/27199, WO 98/07726, WO 97/34895, WO 96/31510, WO 98/14449, WO 98/14450, WO 98/14451, WO 95/09847, WO 97/19065, WO 98/17662, WO 99/35146, WO 99/35132, WO 99/07701, and WO 92/20642; European Patent Application Nos. EP 520722, EP 566226, EP 787772, EP 837063, and EP 682027; U.S. Pat. Nos. 5,747,498, 5,789,427, 5,650,415, and 5,656,643; and German Patent Application No. DE 19629652. Additional non-limiting examples of low molecular weight EGFR kinase inhibitors include any of the EGFR kinase inhibitors described in Traxler, P., 1998, Exp. Opin. Ther. Patents 8(12):1599-1625.

Specific preferred examples of low molecular weight EGFR kinase inhibitors that can be used according to the present invention include [6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine (also known as OSI-774, erlotinib, or TARCEVA® (erlotinib HCl); OSI Pharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; International Patent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997) Cancer Res. 57:4838-4848); CI-1033 (formerly known as PD183805; Pfizer) (Sherwood et al., 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780 (Pfizer); AG-1478 (University of California); CGP-59326 (Novartis); PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); and gefitinib (also known as ZD1839 or IRESSA™; Astrazeneca) (Woodburn et al., 1997, Proc. Am. Assoc. Cancer Res. 38:633). A particularly preferred low molecular weight EGFR kinase inhibitor that can be used according to the present invention is [6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine (i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HCl, TARCEVA®), or other salt forms (e.g. erlotinib mesylate).

EGFR kinase inhibitors also include, for example multi-kinase inhibitors that have activity on EGFR kinase, i.e. inhibitors that inhibit EGFR kinase and one or more additional kinases. Examples of such compounds include the EGFR and HER2 inhibitor CI-1033 (formerly known as PD183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (Boehringer Ingelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); the VEGF-R2 and EGFR inhibitor ZD6474 (also known as ZACTIMA™; AstraZeneca Pharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626 (Bristol-Myers Squibb).

Antibody-based EGFR kinase inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by its natural ligand. Non-limiting examples of antibody-based EGFR kinase inhibitors include those described in Modjtahedi, H., et al., 1993, Br. J. Cancer 67:247-253; Teramoto, T., et al., 1996, Cancer 77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318; Huang, S. M., et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang, X., et al., 1999, Cancer Res. 59:1236-1243. Thus, the EGFR kinase inhibitor can be the monoclonal antibody Mab E7.6.3 (Yang, X. D. et al. (1999) Cancer Res. 59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or an antibody or antibody fragment having the binding specificity thereof. Suitable monoclonal antibody EGFR kinase inhibitors include, but are not limited to, IMC-C225 (also known as cetuximab or ERBITUX™; Imclone Systems), ABX-EGF (Abgenix), EMD 72000 (Merck KgaA, Darmstadt), RH3 (York Medical Bioscience Inc.), and MDX-447 (Medarex/Merck KgaA).

EGFR kinase inhibitors for use in the present invention can alternatively be peptide or RNA aptamers. Such aptamers can for example interact with the extracellular or intracellular domains of EGFR to inhibit EGFR kinase activity in cells. An aptamer that interacts with the extracellular domain is preferred as it would not be necessary for such an aptamer to cross the plasma membrane of the target cell. An aptamer could also interact with the ligand for EGFR (e.g. EGF, TGF-α), such that its ability to activate EGFR is inhibited. Methods for selecting an appropriate aptamer are well known in the art. Such methods have been used to select both peptide and RNA aptamers that interact with and inhibit EGFR family members (e.g. see Buerger, C. et al. et al. (2003) J. Biol. Chem. 278:37610-37621; Chen, C-H. B. et al. (2003) Proc. Natl. Acad. Sci. 100:9226-9231; Buerger, C. and Groner, B. (2003) J. Cancer Res. Clin. Oncol. 129(12):669-675. Epub 2003 Sep. 11.).

EGFR kinase inhibitors for use in the present invention can alternatively be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of EGFR mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of EGFR kinase protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding EGFR can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as EGFR kinase inhibitors for use in the present invention. EGFR gene expression can be reduced by contacting the tumor, subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that expression of EGFR is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschi, T., et al. (1999) Genes Dev. 13(24):3191-3197; Elbashir, S. M. et al. (2001) Nature 411:494-498; Hannon, G. J. (2002) Nature 418:244-251; McManus, M. T. and Sharp, P. A. (2002) Nature Reviews Genetics 3:737-747; Bremmelkamp, T. R. et al. (2002) Science 296:550-553; U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as EGFR kinase inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of EGFR mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as EGFR kinase inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

As used herein, the term “IGF-1R kinase inhibitor” refers to any IGF-1R kinase inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the IGF-1 receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to IGF-1R of its natural ligand. Such IGF-1R kinase inhibitors include any agent that can block IGF-1R activation or any of the downstream biological effects of IGF-1R activation that are relevant to treating cancer in a patient. Such an inhibitor can act by binding directly to the intracellular domain of the receptor and inhibiting its kinase activity. Alternatively, such an inhibitor can act by occupying the ligand binding site or a portion thereof of the IGF-1 receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced. Alternatively, such an inhibitor can act by modulating the dimerization of IGF-1R polypeptides, or interaction of IGF-1R polypeptide with other proteins, or enhance ubiquitination and endocytotic degradation of IGF-1R. An IGF-1R kinase inhibitor can also act by reducing the amount of IGF-1 available to activate IGF-1R, by for example antagonizing the binding of IGF-1 to its receptor, by reducing the level of IGF-1, or by promoting the association of IGF-1 with proteins other than IGF-1R such as IGF binding proteins (e.g. IGFBP3). IGF-1R kinase inhibitors include but are not limited to low molecular weight inhibitors, antibodies or antibody fragments, antisense constructs, small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), and ribozymes. In a preferred embodiment, the IGF-1R kinase inhibitor is a small organic molecule or an antibody that binds specifically to the human IGF-1R.

IGF-1R kinase inhibitors include, for example imidazopyrazine IGF-1R kinase inhibitors, azabicyclic amine inhibitors, quinazoline IGF-1R kinase inhibitors, pyrido-pyrimidine IGF-1R kinase inhibitors, pyrimido-pyrimidine IGF-1R kinase inhibitors, pyrrolo-pyrimidine IGF-1R kinase inhibitors, pyrazolo-pyrimidine IGF-1R kinase inhibitors, phenylamino-pyrimidine IGF-1R kinase inhibitors, oxindole IGF-1R kinase inhibitors, indolocarbazole IGF-1R kinase inhibitors, phthalazine IGF-1R kinase inhibitors, isoflavone IGF-1R kinase inhibitors, quinalone IGF-1R kinase inhibitors, and tyrphostin IGF-1R kinase inhibitors, and all pharmaceutically acceptable salts and solvates of such IGF-1R kinase inhibitors.

Examples of IGF-1R kinase inhibitors include those in International Patent Publication No. WO 05/097800, that describes azabicyclic amine derivatives, International Patent Publication No. WO 05/037836, that describes imidazopyrazine IGF-1R kinase inhibitors, International Patent Publication Nos. WO 03/018021 and WO 03/018022, that describe pyrimidines for treating IGF-1R related disorders, International Patent Publication Nos. WO 02/102804 and WO 02/102805, that describe cyclolignans and cyclolignans as IGF-1R inhibitors, International Patent Publication No. WO 02/092599, that describes pyrrolopyrimidines for the treatment of a disease which responds to an inhibition of the IGF-1R tyrosine kinase, International Patent Publication No. WO 01/72751, that describes pyrrolopyrimidines as tyrosine kinase inhibitors, and in International Patent Publication No. WO 00/71129, that describes pyrrolotriazine inhibitors of kinases, and in International Patent Publication No. WO 97/28161, that describes pyrrolo[2,3-d]pyrimidines and their use as tyrosine kinase inhibitors, Parrizas, et al., which describes tyrphostins with in vitro and in vivo IGF-1R inhibitory activity (Endocrinology, 138:1427-1433 (1997)), International Patent Publication No. WO 00/35455, that describes heteroaryl-aryl ureas as IGF-1R inhibitors, International Patent Publication No. WO 03/048133, that describes pyrimidine derivatives as modulators of IGF-1R, International Patent Publication No. WO 03/024967, WO 03/035614, WO 03/035615, WO 03/035616, and WO 03/035619, that describe chemical compounds with inhibitory effects towards kinase proteins, International Patent Publication No. WO 03/068265, that describes methods and compositions for treating hyperproliferative conditions, International Patent Publication No. WO 00/17203, that describes pyrrolopyrimidines as protein kinase inhibitors, Japanese Patent Publication No. JP 07/133,280, that describes a cephem compound, its production and antimicrobial composition, Albert, A. et al., Journal of the Chemical Society, 11: 1540-1547 (1970), which describes pteridine studies and pteridines unsubstituted in the 4-position, and A. Albert et al., Chem. Biol. Pteridines Proc. Int. Symp., 4th, 4: 1-5 (1969) which describes a synthesis of pteridines (unsubstituted in the 4-position) from pyrazines, via 3-4-dihydropteridines.

Additional, specific examples of IGF-1R kinase inhibitors that can be used according to the present invention include h7C10 (Centre de Recherche Pierre Fabre), an IGF-1 antagonist; EM-164 (ImmunoGen Inc.), an IGF-1R modulator; CP-751871 (Pfizer Inc.), an IGF-1 antagonist; lanreotide (Ipsen), an IGF-1 antagonist; IGF-1R oligonucleotides (Lynx Therapeutics Inc.); IGF-1 oligonucleotides (National Cancer Institute); IGF-1R protein-tyrosine kinase inhibitors in development by Novartis (e.g. NVP-AEW541, Garcia-Echeverria, C. et al. (2004) Cancer Cell 5:231-239; or NVP-ADW742, Mitsiades, C. S. et al. (2004) Cancer Cell 5:221-230); IGF-1R protein-tyrosine kinase inhibitors (Ontogen Corp); OSI-906 (OSI Pharmaceuticals); AG-1024 (Camirand, A. et al. (2005) Breast Cancer Research 7:R570-R579 (DOI 10.1186/bcr1028); Camirand, A. and Pollak, M. (2004) Brit. J. Cancer 90:1825-1829; Pfizer Inc.), an IGF-1 antagonist; the tyrphostins AG-538 and I-OMe-AG 538; BMS-536924, a small molecule inhibitor of IGF-1R; PNU-145156E (Pharmacia & Upjohn SpA), an IGF-1 antagonist; BMS 536924, a dual IGF-1R and IR kinase inhibitor (Bristol-Myers Squibb); AEW541 (Novartis); GSK621659A (Glaxo Smith-Kline); INSM-18 (Insmed); and XL-228 (Exelixis).

Antibody-based IGF-1R kinase inhibitors include any anti-IGF-1R antibody or antibody fragment that can partially or completely block IGF-1R activation by its natural ligand. Antibody-based IGF-1R kinase inhibitors also include any anti-IGF-1 antibody or antibody fragment that can partially or completely block IGF-1R activation. Non-limiting examples of antibody-based IGF-1R kinase inhibitors include those described in Larsson, O. et al (2005) Brit. J. Cancer 92:2097-2101 and Ibrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res. 11:944s-950s; or being developed by Imclone (e.g. IMC-A12), or AMG-479, an anti-IGF-1R antibody (Amgen); R1507, an anti-IGF-1R antibody (Genmab/Roche); AVE-1642, an anti-IGF-1R antibody (Immunogen/Sanofi-Aventis); MK 0646 or h7C10, an anti-IGF-1R antibody (Merck); or antibodies being develop by Schering-Plough Research Institute (e.g. SCH 717454 or 19D12; or as described in US Patent Application Publication Nos. US 2005/0136063 A1 and US 2004/0018191 A1). The IGF-1R kinase inhibitor can be a monoclonal antibody, or an antibody or antibody fragment having the binding specificity thereof.

As used herein, the term “PDGFR kinase inhibitor” refers to any PDGFR kinase inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the PDGF receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to PDGFR of its natural ligand. Such PDGFR kinase inhibitors include any agent that can block PDGFR activation or any of the downstream biological effects of PDGFR activation that are relevant to treating cancer in a patient. Such an inhibitor can act by binding directly to the intracellular domain of the receptor and inhibiting its kinase activity. Alternatively, such an inhibitor can act by occupying the ligand binding site or a portion thereof of the PDGF receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced. Alternatively, such an inhibitor can act by modulating the dimerization of PDGFR polypeptides, or interaction of PDGFR polypeptide with other proteins, or enhance ubiquitination and endocytotic degradation of PDGFR. PDGFR kinase inhibitors include but are not limited to low molecular weight inhibitors, antibodies or antibody fragments, antisense constructs, small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), and ribozymes. PDGFR kinase inhibitors include anti-PDGF or anti-PDGFR aptamers, anti-PDGF or anti-PDGFR antibodies, or soluble PDGF receptor decoys that prevent binding of a PDGF to its cognate receptor. In a preferred embodiment, the PDGFR kinase inhibitor is a small organic molecule or an antibody that binds specifically to the human PDGFR. The ability of a compound or agent to serve as a PDGFR kinase inhibitor may be determined according to the methods known in art and, further, as set forth in, e.g., Dai et al., (2001) Genes & Dev. 15: 1913-25; Zippel, et al., (1989) Eur. J. Cell Biol. 50(2):428-34; and Zwiller, et al., (1991) Oncogene 6: 219-21.

The invention includes PDGFR kinase inhibitors known in the art as well as those supported below and any and all equivalents that are within the scope of ordinary skill to create. For example, inhibitory antibodies directed against PDGF are known in the art, e.g., those described in U.S. Pat. Nos. 5,976,534, 5,833,986, 5,817,310, 5,882,644, 5,662,904, 5,620,687, 5,468,468, and PCT WO 2003/025019, the contents of which are incorporated by reference in their entirety. In addition, the invention includes N-phenyl-2-pyrimidine-amine derivatives that are PDGFR kinase inhibitors, such as those disclosed in U.S. Pat. No. 5,521,184, as well as WO2003/013541, WO2003/078404, WO2003/099771, WO2003/015282, and WO2004/05282 which are hereby incorporated in their entirety by reference.

Small molecules that block the action of PDGF are known in the art, e.g., those described in U.S. Pat. Nos. or Published Application Nos. 6,528,526 (PDGFR tyrosine kinase inhibitors), 6,524,347 (PDGFR tyrosine kinase inhibitors), 6,482,834 (PDGFR tyrosine kinase inhibitors), 6,472,391 (PDGFR tyrosine kinase inhibitors), 6,949,563, 6,696,434, 6,331,555, 6,251,905, 6,245,760, 6,207,667, 5,990,141, 5,700,822, 5,618,837, 5,731,326, and 2005/0154014, and International Published Application Nos. WO 2005/021531, WO 2005/021544, and WO 2005/021537, the contents of which are incorporated by reference in their entirety.

Proteins and polypeptides that block the action of PDGF are known in the art, e.g., those described in U.S. Pat. Nos. 6,350,731 (PDGF peptide analogs), 5,952,304, the contents of which are incorporated by reference in their entirety.

Bis mono- and bicyclic aryl and heteroaryl compounds which inhibit EGF and/or PDGF receptor tyrosine kinase are known in the art, e.g., those described in, e.g. U.S. Pat. Nos. 5,476,851, 5,480,883, 5,656,643, 5,795,889, and 6,057,320, the contents of which are incorporated by reference in their entirety.

Antisense oligonucleotides for the inhibition of PDGF are known in the art, e.g., those described in U.S. Pat. Nos. 5,869,462, and 5,821,234, the contents of each of which are incorporated by reference in their entirety.

Aptamers (also known as nucleic acid ligands) for the inhibition of PDGF are known in the art, e.g., those described in, e.g., U.S. Pat. Nos. 6,582,918, 6,229,002, 6,207,816, 5,668,264, 5,674,685, and 5,723,594, the contents of each of which are incorporated by reference in their entirety.

Other compounds for inhibiting PDGF known in the art include those described in U.S. Pat. Nos. 5,238,950, 5,418,135, 5,674,892, 5,693,610, 5,700,822, 5,700,823, 5,728,726, 5,795,910, 5,817,310, 5,872,218, 5,932,580, 5,932,602, 5,958,959, 5,990,141, 6,358,954, 6,537,988 and 6,673,798, the contents of each of which are incorporated by reference in their entirety.

A number of types of tyrosine kinase inhibitors that are selective for tyrosine kinase receptor enzymes such as PDGFR are known (see, e.g., Spada and Myers ((1995) Exp. Opin. Ther. Patents, 5: 805) and Bridges ((1995) Exp. Opin. Ther. Patents, 5: 1245). Additionally Law and Lydon have summarized the anticancer potential of tyrosine kinase inhibitors ((1996) Emerging Drugs: The Prospect For Improved Medicines, 241-260). For example, U.S. Pat. No. 6,528,526 describes substituted quinoxaline compounds that selectively inhibit platelet-derived growth factor-receptor (PDGFR) tyrosine kinase activity. The known inhibitors of PDGFR tyrosine kinase activity includes quinoline-based inhibitors reported by Maguire et al., ((1994) J. Med. Chem., 37: 2129), and by Dolle, et al., ((1994) J. Med. Chem., 37: 2627). A class of phenylamino-pyrimidine-based inhibitors was recently reported by Traxler, et al., in EP 564409 and by Zimmerman et al., ((1996) Biorg. Med. Chem. Lett., 6: 1221-1226) and by Buchdunger, et al., ((1995) Proc. Nat. Acad. Sci. (USA), 92: 2558). Quinazoline derivatives that are useful in inhibiting PDGF receptor tyrosine kinase activity include bismono- and bicyclic aryl compounds and heteroaryl compounds (see, e.g., WO 92/20642), quinoxaline derivatives (see (1994) Cancer Res., 54: 6106-6114), pyrimidine derivatives (Japanese Published Patent Application No. 87834/94) and dimethoxyquinoline derivatives (see Abstracts of the 116th Annual Meeting of the Pharmaceutical Society of Japan (Kanazawa), (1996), 2, p. 275, 29(C2) 15-2).

Specific preferred examples of low molecular weight PDGFR kinase inhibitors that can be used according to the present invention include Imatinib (GLEEVEC®; Novartis); SU-12248 (sunitib malate, SUTENT®; Pfizer); Dasatinib (SPRYCEL®; BMS; also known as BMS-354825); Sorafenib (NEXAVAR®; Bayer; also known as Bay-43-9006); AG-13736 (Axitinib; Pfizer); RPR127963 (Sanofi-Aventis); CP-868596 (Pfizer/OSI Pharmaceuticals); MLN-518 (tandutinib; Millennium Pharmaceuticals); AMG-706 (Motesanib; Amgen); ARAVA® (leflunomide; Sanofi-Aventis; also known as SU101), and OSI-930 (OSI Pharmaceuticals); Additional preferred examples of low molecular weight PDGFR kinase inhibitors that are also FGFR kinase inhibitors that can be used according to the present invention include XL-999 (Exelixis); SU6668 (Pfizer); CHIR-258/TKI-258 (Chiron); RO4383596 (Hoffmann-La Roche) and BIBF-1120 (Boehringer Ingelheim).

As used herein, the term “FGFR kinase inhibitor” refers to any FGFR kinase inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the FGF receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to FGFR of its natural ligand. Such FGFR kinase inhibitors include any agent that can block FGFR activation or any of the downstream biological effects of FGFR activation that are relevant to treating cancer in a patient. Such an inhibitor can act by binding directly to the intracellular domain of the receptor and inhibiting its kinase activity. Alternatively, such an inhibitor can act by occupying the ligand binding site or a portion thereof of the FGF receptor, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced. Alternatively, such an inhibitor can act by modulating the dimerization of FGFR polypeptides, or interaction of FGFR polypeptide with other proteins, or enhance ubiquitination and endocytotic degradation of FGFR. FGFR kinase inhibitors include but are not limited to low molecular weight inhibitors, antibodies or antibody fragments, antisense constructs, small inhibitory RNAs (i.e. RNA interference by dsRNA; RNAi), and ribozymes. FGFR kinase inhibitors include anti-FGF or anti-FGFR aptamers, anti-FGF or anti-FGFR antibodies, or soluble FGFR receptor decoys that prevent binding of a FGFR to its cognate receptor. In a preferred embodiment, the FGFR kinase inhibitor is a small organic molecule or an antibody that binds specifically to the human FGFR. Anti-FGFR antibodies include FR1-H7 (FGFR-1) and FR3-D11 (FGFR-3) (Imclone Systems, Inc.).

FGFR kinase inhibitors also include compounds that inhibit FGFR signal transduction by affecting the ability of heparan sulfate proteoglycans to modulate FGFR activity. Heparan sulfate proteoglycans in the extracellular matrix can mediate the actions of FGF, e.g., protection from proteolysis, localization, storage, and internalization of growth factors (Faham, S. et al. (1998) Curr. Opin. Struct. Biol., 8:578-586), and may serve as low affinity FGF receptors that act to present FGF to its cognate FGFR, and/or to facilitate receptor oligomerization (Galzie, Z. et al. (1997) Biochem. Cell. Biol., 75:669-685).

The invention includes FGFR kinase inhibitors known in the art (e.g. PD173074) as well as those supported below and any and all equivalents that are within the scope of ordinary skill to create.

Examples of chemicals that may antagonize FGF action, and can thus be used as FGFR kinase inhibitors in the methods described herein, include suramin, structural analogs of suramin, pentosan polysulfate, scopolamine, angiostatin, sprouty, estradiol, carboxymethylbenzylamine dextran (CMDB7), suradista, insulin-like growth factor binding protein-3, ethanol, heparin (e.g., 6-O-desulfated heparin), low molecular weight heparin, protamine sulfate, cyclosporin A, or RNA ligands for bFGF.

Other agents or compounds for inhibiting FGFR kinase known in the art include those described in U.S. Pat. Nos. 7,151,176 (Bristol-Myers Squibb Company; Pyrrolotriazine compounds); 7,102,002 (Bristol-Myers Squibb Company; pyrrolotriazine compounds); 5,132,408 (Salk Institute; peptide FGF antagonists); and 5,945,422 (Warner-Lambert Company; 2-amino-substituted pyrido[2,3-d]pyrimidines); U.S. published Patent application Nos. 2005/0256154 (4-amino-thieno[3,2-c]pyridine-7-carboxylic acid amide compounds); and 2004/0204427 (pyrimidino compounds); and published International Patent Applications WO-2007019884 (Merck Patent GmbH; N-(3-pyrazolyl)-N′-4-(4-pyridinyloxy)phenyl)urea compounds); WO-2007009773 (Novartis AG; pyrazolo[1,5-a]pyrimidin-7-yl amine derivatives); WO-2007014123 (Five Prime Therapeutics, Inc.; FGFR fusion proteins); WO-2006134989 (Kyowa Hakko Kogyo Co., Ltd.; nitrogenous heterocycle compounds); WO-2006112479 (Kyowa Hakko Kogyo Co., Ltd.; azaheterocycles); WO-2006108482 (Merck Patent GmbH; 9-(4-ureidophenyl)purine compounds); WO-2006105844 (Merck Patent GmbH; N-(3-pyrazolyl)-N′-4-(4-pyridinyloxy)phenyl)urea compounds); WO-2006094600 (Merck Patent GmbH; tetrahydropyrroloquinoline derivatives); WO-2006050800 (Merck Patent GmbH; N,N′-diarylurea derivatives); WO-2006050779 (Merck Patent GmbH; N,N′-diarylurea derivatives); WO-2006042599 (Merck Patent GmbH; phenylurea derivatives); WO-2005066211 (Five Prime Therapeutics, Inc.; anti-FGFR antibodies); WO-2005054246 (Merck Patent GmbH; heterocyclyl amines); WO-2005028448 (Merck Patent GmbH; 2-amino-1-benzyl-substituted benzimidazole derivatives); WO-2005011597 (Irm Llc; substituted heterocyclic derivatives); WO-2004093812 (Irm Llc/Scripps; 6-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives); WO-2004046152 (F. Hoffmann La Roche A G; pyrimido[4,5-e]oxadiazine derivatives); WO-2004041822 (F. Hoffmann La Roche A G; pyrimido[4,5-d]pyrimidine derivatives); WO-2004018472 (F. Hoffmann La Roche AG; pyrimido[4,5-d]pyrimidine derivatives); WO-2004013145 (Bristol-Myers Squibb Company; pyrrolotriazine derivatives); WO-2004009784 (Bristol-Myers Squibb Company; pyrrolo[2,1-f][1,2,4]triazin-6-yl compounds); WO-2004009601 (Bristol-Myers Squibb Company; azaindole compounds); WO-2004001059 (Bristol-Myers Squibb Company; heterocyclic derivatives); WO-02102972 (Prochon Biotech Ltd./Morphosys AG; anti-FGFR antibodies); WO-02102973 (Prochon Biotech Ltd.; anti-FGFR antibodies); WO-00212238 (Warner-Lambert Company; 2-(pyridin-4-ylamino)-6-dialkoxyphenyl-pyrido[2,3-d]pyrimidin-7-one derivatives); WO-00170977 (Amgen, Inc.; FGFR-L and derivatives); WO-00132653 (Cephalon, Inc.; pyrazolone derivatives); WO-00046380 (Chiron Corporation; FGFR-Ig fusion proteins); and WO-00015781 (Eli Lilly; polypeptides related to the human SPROUTY-1 protein).

Specific preferred examples of low molecular weight FGFR kinase inhibitors that can be used according to the present invention include RO-4396686 (Hoffmann-La Roche); CHIR-258 (Chiron; also known as TKI-258); PD 173074 (Pfizer); PD 166866 (Pfizer); ENK-834 and ENK-835 (both Enkam Pharmaceuticals A/S); and SU5402 (Pfizer). Additional preferred examples of low molecular weight FGFR kinase inhibitors that are also PDGFR kinase inhibitors that can be used according to the present invention include XL-999 (Exelixis); SU6668 (Pfizer); CHIR-258/TKI-258 (Chiron); R04383596 (Hoffmann-La Roche), and BIBF-1120 (Boehringer Ingelheim).

The present invention also provides a method of identifying an agent that inhibits epithelial cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial cell line CFPAC-1 with a test agent to be screened, contacting the sample with a single protein ligand preparation that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells, determining whether the test agent inhibits the epithelial cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample cells to the level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits cells from undergoing an epithelial to mesenchymal transition. Agents that inhibit epithelial cells from undergoing an epithelial to mesenchymal transition are useful for the treatment of fibrotic disorders resulting in part from EMT transitions, including but not limited to renal fibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agent that inhibits cells that have undergone an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition. Agents that inhibit mesenchymal-like cells are useful for the treatment of fibrotic disorders resulting in part from EMT transitions, including but not limited to renal fibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4. An alternative embodiment of this method comprises, after the step of determining whether the test agent inhibits the growth of cells that have undergone an epithelial to mesenchymal transition, the additional steps of determining whether an agent that inhibits mesenchymal-like CFPAC-1 tumor cell growth, also inhibits epithelial CFPAC-1 tumor cell growth, and thus determining whether it is an agent that specifically inhibits the growth of cells that have undergone an epithelial to mesenchymal transition. In an embodiment of the above methods, an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition is determined to do so by stimulating apoptosis of said cells. In another embodiment of the above methods, an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition is determined to do so by inhibiting proliferation of said cells.

The present invention also provides a method of identifying an agent that stimulates mesenchymal-like cells to undergo a mesenchymal to epithelial transition, comprising contacting a sample of cells of the epithelial cell line CFPAC-1 with a single protein ligand preparation to induce an epithelial-to-mesenchymal transition in the CFPAC-1 cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like cells to undergo a mesenchymal to epithelial transition. Agents that stimulate mesenchymal-like cells to undergo a mesenchymal to epithelial transition are useful for the treatment of fibrotic disorders resulting in part from EMT transitions, including but not limited to renal fibrosis, hepatic fibrosis, pulmonary fibrosis, and mesotheliomas. In one embodiment, the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; and BMP4.

The present invention also provides a method of identifying an agent that inhibits cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a test agent to be screened, contacting the sample with a compound that induces the expression of said protein that stimulates an epithelial to mesenchymal transition in the engineered CFPAC-1 cells, determining whether the test agent inhibits the cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample cells to the level of the same biomarker in an identical sample of engineered CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits cells from undergoing an epithelial to mesenchymal transition.

The present invention also provides a method of identifying an agent that inhibits cells that have undergone an epithelial to mesenchymal transition, comprising: contacting a sample of cells of the epithelial cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a compound that induces the expression of said protein such that an epithelial-to-mesenchymal transition is induced in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like CFPAC-1 cell growth, and thus determining whether it is an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition. An alternative embodiment of this method comprises, after the step of determining whether the test agent inhibits the growth of cells that have undergone an epithelial to mesenchymal transition, the additional steps of determining whether an agent that inhibits mesenchymal-like CFPAC-1 cell growth, also inhibits epithelial CFPAC-1 cell growth, and thus determining whether it is an agent that specifically inhibits the growth of cells that have undergone an epithelial to mesenchymal transition. In an embodiment of the above methods, an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition is determined to do so by stimulating apoptosis of said cells. In another embodiment of the above methods, an agent that inhibits the growth of cells that have undergone an epithelial to mesenchymal transition is determined to do so by inhibiting proliferation of said cells.

The present invention also provides a method of identifying an agent that stimulates mesenchymal-like cells to undergo a mesenchymal to epithelial transition, comprising contacting a sample of cells of the epithelial cell line CFPAC-1, which have been engineered to inducibly express a protein that stimulates an epithelial to mesenchymal transition in CFPAC-1 cells, with a compound that induces the expression of said protein such that an epithelial-to-mesenchymal transition is induced in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates the mesenchymal-like CFPAC-1 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample cells to the level of the same biomarker in an identical sample of mesenchymal-like CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like cells to undergo a mesenchymal to epithelial transition.

This invention will be better understood from the Experimental Details that follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter, and are not to be considered in any way limited thereto.

Experimental Details:

Introduction

Models for the identification of targeted anti-cancer agents and the rational design of specific combinations of anti-cancer agents are clearly needed to advance such agents to clinical testing. Here we describe model systems for both epithelial and mesenchymal tumor cell components. The ability to target both epithelial and mesenchymal cell types within tumors will be critical to a therapeutic impact on long term patient survival. The models described enable evaluation of anti-cancer compounds, antibodies, aptamers and other therapeutic nucleic acids toward the tumors cell types in vitro and in animal models.

Materials and Methods

Cell Culture & Treatments

The CFPAC1 cell line was cultured in DMEM media (Gibco #11960) supplemented with 10% FBS (Sigma), 2 mM L Glutamine (Gibco), at 37° C., 5% CO₂. For 7 day treatments, cells were seeded on tissue culture plastic in complete growth media and allowed to attach overnight at cell densities that would provide approximately 60-80% confluence at end of experiment. Ligand and/or inhibitor was added the following day (Day 0) in complete growth media and incubated at 37° C., 5% CO₂. Ligand source and concentrations used are as follows: [1] From R&D systems, SDF-1α (stromal derived factor-1 alpha) at 100 ng/ml, TRANCE (TNF-related activation induced cytokine) at 50 ng/ml, FGF1 (fibroblast growth factor acidic) at 10 ng/ml, FGF2 (fibroblast growth factor basic) at 10 ng/ml, BMP4 (bone morphogenetic protein 4) at 100 ng/ml, BMP7 (bone morphogenetic protein 7) at 100 ng/ml, IGF2 (insulin-like growth factor 2) at 100 ng/ml, OSM (Oncostatin M) at 100 ng/ml, NRG1 (Neuregulin-1) at 50 ng/ml, PDGFaa (Platelet derived growth factor a) at 50 ng/ml, PDGFbb (platelet derive growth factor b) at 30 ng/ml, Ang2 (Angiopoeitin 2) at 400 ng/ml, amphiregulin at 100 ng/ml, HMGB 1 (Human high-mobility group box 1 protein) at 50 ng/ml; [2] From Genscript, LIF (Leukemia inhibitory factor) at 20 ng/ml, GM-CSF (Granulocyte macrophage colony stimulating factor) at 25 ng/ml, M-CSF (Macrophage colony stimulating factor) at 50 ng/ml; [3] From Anaspec, PAR1 (Protease activated receptor 1 agonist) at 100 μM; PAR2 (Protease activated receptor 2 agonist; peptide SLIGKV-NH₂, Cat. No. 60217-1) at 100 μM; PAR4 (Protease activated receptor δ agonist; peptide AYPGKF-NH₂, Cat. No. 60218-1) at 100 μM; [4] From Peprotech, HGF (Hepatocyte Growth Factor) at 100 ng/ml; IL-31 (Interleukin-31) at 50 ng/ml, IL-33 (Interleukin-33) at 50 ng/ml, IL1-a (Interleukin-1 alpha) at 10 ng/ml, CTGF (Connective tissue growth factor) at 20 ng/ml, WISP1 (Wnt-1 inducible signaling pathway protein) at 20 ng/ml, PTHrP (Polypeptide hormone-related protein) at 50 ng/ml, MCP1 (monocyte chemotactic protein-1) at 50 ng/ml; and [5] from Sigma, Fulvestrant at 1 μM. Fresh medium containing ligand and/or inhibitor was added 3-4 days after initial dose, and at Day 7 cells were harvested for western blot analysis.

Pharmacological Inhibitors

The following inhibitors were purchased from EMD Biosciences: JAK inhibitor, 1 (catalog #420099) used at 0.25 μM and MEK inhibitor 1 (catalog #444937) used at 3 μM.

Western Blotting and Antibodies

Cells were harvested by washing once with PBS, and lysing on ice for 10 minutes with scraping (using a Cell Lifter, Corning Inc. #3008) in ice cold RIPA buffer (Sigma R0278) containing Protease inhibitor (Sigma P8340), Phosphatase inhibitors (Sigma P2850, P5726) and 200 μM Sodium Vanadate. Supernatents were micro-centrifuged at max speed, 4° C. for 10 minutes. Protein content was quantitated using the BCA method (Pierce #23225) and samples were normalized to total protein in Laemli buffer and heated at 100° C. for 10 minutes. SDS PAGE was run using a Bis-Tris gel system (Invitrogen NuPAGE) with MOPS buffer (Invitrogen NP0001), transferred onto nitrocellulose using the iBLOT system (Invitrogen) and blocked in PBS-T (0.1% Tween 20), 5% non-fat milk or BSA in guidance with primary Ab supplier requirements. All primary antibodies were incubated overnight at 4° C. in PBS-T (0.1% Tween 20) in either 5% non-fat milk or BSA, diluted to the following working dilutions: anti E Cadherin (Santa Cruz #sc-21791) 1:500 dilution; anti Vimentin (BD Biosciences #550513) 1:2000 dilution;); anti GAPDH HRP conjugate (Santa Cruz sc-25778) 1:1000 dilution; anti β-Actin (Sigma, A5441) 1:10,000 dilution. Secondary HRP labeled antibodies used were: anti Rabbit IgG HRP (Amersham NA934) 1:5000 dilution; anti Mouse IgG HRP (Sigma A2304) 1:2000 dilution. Membranes were visualized using the Supersignal ELISA Femto Maximum Sensitivity Substrate (Pierce).

Immunofluorescence/Confocal Microscopy

Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences #15710)/PBS (Gibco#14190) for 10 minutes at room temp, washed with PBS and permeabilized with 0.1% Triton X100 (Sigma P1379) in PBS for 10 minutes at room temp. Cells were blocked with 3% BSA/PBS for at least 15 minutes then incubated with primary antibody diluted in blocking buffer for a minimum of two hours at room temperature. Anti E-Cadherin was used at a 1:75 dilution and anti Vimentin, (Millipore #AB5733) was used at a 1:2000 dilution. Cells were washed, incubated with fluorescently-labeled secondary antibodies for one hour, followed by a 5 minute TO-PRO3 nuclear counterstain (Invitrogen #T3605) at room temperature, washed and mounted on slides with Pro-Long Gold anti-fade reagent (Invitrogen #P36934). Secondary fluorescent antibodies used were: anti mouse 488 nm (Invitrogen #A11029) 1:600 dilution; anti chicken TRITC (Chemicon #AP194R) 1:50 dilution. Stained cells were captured on a Leica DMRXE microscope/SP2 scanner using Leica Confocal Software (LCS).

Chemotaxis/Invasion Assay

Cells were seeded and stimulated as described in the ligand treatment section above, over a 7 day period. On the 7^(th) day cells were serum-starved in the presence of ligand for 24 hours prior to plating in modified Boyden chambers (Trevigen Cultrex #3458-096-K). Cells were plated in serum-free medium in the upper chamber at 50,000 cells/well, with 3× concentration of ligand and 10% FBS in the lower chamber as a chemo-attractant. 0% FBS was used as a control for comparison. For migration assays, membranes were left uncoated; for invasion assays, membranes were coated with type IV collagen or basement membrane extract (provided with kit) 24 hours prior to seeding. After 24 (migration) or 48 (invasion) hours, cells that had traversed the membrane and attached to the underside of the membrane were quantified using calcein-AM read on a fluorescent plate reader (Wallac).

MET/EMT Reversion Experiment

Cells were cultured in complete medium with and without ligand for 14 days, during which time the media and ligand treatment was replaced every 3-4 days. 3 Days prior to the first time point (Day 14), cells were trypsinised, counted and seeded into plastic 35 mm dishes at appropriate seeding densities for the first 3 collection time points. 3 Days prior to the last of the 3 collection time points, the procedure was repeated for the second 3 collection time points, and so on until all time points were collected. Ligand was removed from all cultures at Day 14. At the time of harvest, cells were harvested as described in the western blotting section above. All samples were micro-centrifuged and stored at −80° C. until the end of the experiment, at which time all samples were processed for western blotting.

2D Immunofluorescence and Confocal Microscopy

Cells were plated on coverslips and stimulated with ligand the following day (day 0). On day 4, medium and ligands were refreshed. After 7 days, the cells were fixed in 4% paraformaldehyde (EMS#15701)/PBS (Gibco #14190) for 10 minutes at room temperature, and then permeabilized in 0.1% Triton X-100 (Sigma #P1379)/PBS for 10 minutes. Cells were blocked in 3% BSA/PBS for one hour and incubated in primary antibody solution (E-cadherin, Santa Cruz #sc21791 and Vimentin, Chemicon #AB5733 in blocking buffer) for two hours at room temperature. Cells were washed in PBS and incubated in secondary antibody (Invitrogen #A11029 and Chemicon #AP194R) for one hour followed by nuclear counterstain TO-PRO3 (Invitrogen #T3605). Coverslips were mounted on slides with Pro-Long Gold antifade reagent (Invitrogen P36934). Images were captured on a Leica DMRXE microcope with SP2 scanner using Leica Confocal Software.

Doubling Time:

Cells were plated and treated with ligand for 7 days. On day 7, cells were counted and re-plated on 6 cm cell culture dishes. On days 3, 5, 7, 10 and 12, cells were trypsinized and counted to determine the total number of cells in the dish, in duplicate. Doubling time was calculated using the multiple time point method on the website www.doubling-time.com.

qPCR:

Cells were plated and treated with ligand for one or 7 days. At the time of harvest, cells were trypsinized and snap frozen for later use. Total RNA was isolated using RNAqueous-4 PCR Kit (Ambion, AM1914). For qPCR on tumor material, the tumor samples were homogenized using a small tissue homogenizer (Ultra-Turrax T8 from IKA-Werke) in 175ul RNA lysis buffer. Homogenization was for 1 minute on ice. RNA was isolated using the SV Total RNA Isolation System (Promega Cat #Z3100) according to the manufactures instructions. Samples were DNase treated using the Turbo DNA-Free kit (Ambion, AM1907) and reverse transcribed using Superscript III (Invitrogen, 18080-044) for qPCR analysis. Taqman primers and Locked-Nucleic Acid probes were designed using ProbeFinder software (Universal Probe library, Roche). For mRNA analysis, real time PCR was performed using the ABI 7900HT series PCR machine. Thermocycle conditions were as follows: 50° C. for 2 minutes, 95° C. for 10 minutes, 95° C. for 15 seconds, 50° C. for 10 seconds, 60° C. for 1 minute. Data was collected over 45 cycles and then normalized to GAPDH and further normalized to the untreated control sample.

3D Matrigel Culture:

80 μl of cold Growth Factor Reduced Matrigel (BD Biosciences #354230) was plated per well into 8 well chamber slides (Labtek II, Nunc #154534) and solidified at 37° C. Cells were trypsinized, counted and suspended in complete medium containing 2% Matrigel, to give 5000 cells per 300 μl. 300 μl was aliquoted into each well and incubated at 37° C., 5% CO₂ overnight. Medium was aspirated and replaced with 120 μl per well of medium containing 2% Matrigel and ligand treatments. Cells were grown for 14 days, feeding every 3-4 days. Cultures were imaged by phase contrast.

3D Immuno-Fluorescence and Confocal Imaging:

3D cultures were fixed in 2% paraformaldehyde (Electron Microscopy Sciences #15710)/PBS (Gibco#14190) for 10-20 minutes at room temperature, washed with PBS and permeabilized with 0.1% Triton X100 (Sigma #P1379) in PBS for 20 minutes. Cells were blocked with 3% BSA/PBS for 1 hour then incubated with primary antibody in blocking buffer overnight at room temperature. Dilutions are as follows: anti E-Cadherin (Santa Cruz #sc21791), 1:75; anti Vimentin (Millipore #AB5733), 1:2000. Cells were washed in PBS, and incubated with secondary antibodies overnight at room temperature in the dark (anti-mouse Alexa Fluor 488 nm, Invitrogen #A11029, 1:600; anti-chicken Alexa Fluor 568 nm, Invitrogen #A11041, 1:600). Cells were washed 3×20 minutes in PBS, the third wash containing 5 μM TO-PRO3 (Invitrogen #T3605), and mounted with Pro-Long Gold anti-fade reagent (Invitrogen #P36934). Cells were imaged on a Leica DMRXE microscope/SP2 scanner using Leica Confocal Software (LCS).

In vivo Growth and Immunohistochemistry:

CFPAC 1 cells (1*10e6) were implanted orthotopically into the pancreas of nude mice. The tumors were allowed to establish and the animals sacrificed at 2, 4, and 8 weeks for evaluation of the tumors. Tumors were fixed in Formalin overnight, paraffin embedded and sectioned. Sections were stained using Dako reagents and protocols. For E-Cadherin staining, sections were pretreated with Target Retrieval Solution (#S1699), rinsed with TBS/Tween20 and then blocked first with Peroxidase blocking reagent (#S2001) then with Protein Blocking solution from the Vectastain Elite Rabbit Kit (#PK6106). Sections were rinsed and incubated with primary antibody (E-cadherin CST#3195) diluted at 1:50 in Dako antibody diluent (#S0809) for one hour at room temperature. Sections were rinsed, incubated in Vectastain kit rabbit secondary antibody followed by incubation with Vectastain ABC solution for 30 minutes. Sections were rinsed and developed with DAB+Chromogen, then rinsed, counterstained with Gill's Hematoxylin and mounted. Vimentin staining was done following the same protocol with the Vectastain Elite Goat kit (PK6105) using Chemicon Vimentin #AB5733 chicken polyclonal at 1:6400 dilution, and a solution of rabbit anti-goat and anti-chicken secondary antibody.

Results

EMT Ligand Driven Cell Models

The ability of cell lines to undergo epithelial to mesenchymal transition (EMT) was investigated using different extracellular ligand drivers. A panel of NSCLC, pancreatic, and breast cancer cell lines were screened by treatment of cells for 7 days in presence of respective ligands, either singly or in combination. Initial screening determined the ability of the ligand to cause morphology change, to down-regulate the epithelial marker, e-cadherin or to up-regulate a mesenchymal marker such as vimentin. Potential EMT cell models should up-regulate a mesenchymal marker and display morphology change. More robust models would show a down-regulation of e-cadherin expression. For those models where protein levels of e-cadherin were not substantially down-regulated, it is postulated that e-cadherin is mislocalized.

The CFPAC-1 pancreatic cell line has the propensity to undergo the EMT process with a variety of ligands. FIG. 1 summarizes a proportion of ligands tested that have impact on upregulating vimentin with some ligand treatments giving marked downregulation of E-cadherin.

The following conclusions were made. HGF and OSM induce the most complete EMT in CFPAC-1 cells. Partial EMT was observed with BMP7, IGF2, LIF, PAR2 agonist SLIGKV-NH₂, IL-33, PAR4 agonist AYPGKF-NH₂, CTGF, and BMP4.

Studies to further investigate the role of HGF and OSM to drive CFPAC-1 cells were performed. CFPAC-1 cells were treated with varying concentrations of HGF or OSM for 7 days to determine optimal conditions that would exhibit protein marker changes for E-cadherin and vimentin (FIG. 2). Using optimal conditions of HGF and OSM effects of EMT were monitored for morphology change (FIG. 3), for marker changes b confocal (FIG. 4) and for the ability of cells to increase migration and invasion was investigated (FIG. 5). As shown, cells that were treated with ligand were better able to migrate and invade.

The epithelial to mesenchymal transition process for CFPAC-1 cells can be reversed upon withdrawal of the stimulating ligands, HGF, OSM or with the combination of HGF and OSM (FIG. 6).

The signaling events for the ability of OSM to trigger EMT in CFPAC-1 cells was investigated using inhibitors to the JAK and to MEK to determine the role of the JAK/STAT and MAPK kinase pathway. The JAK inhibitor could block EMT process and the MEK inhibitor was unable to block the EMT process (FIG. 7). The conclusion was made that the JAK/STAT pathway plays a more critical role for ability of CFPAC-1 cells to undergo EMT.

Imaging of EMT In vitro

To characterize EMT in CFPAC 1 cells on the single-cell level, expression of E-cadherin and vimentin in HGF, OSM and HGF+OSM treated cells compared to untreated cells (FIG. 8) was examined. The untreated population showed E-cadherin membrane localization at cell-cell junctions with some cells expressing low levels of vimentin. In cell populations treated with HGF or OSM alone, most cells grew as isolated cells, but some small clusters remained. The majority of cells showed strong vimentin staining with no E-cadherin. In cells that did express E-cadherin, it was localized in the cytoplasm where it is considered non-functional. Cells that were treated with HGF+OSM ligand treatment showed fewer clusters than the single ligand-treated populations, and fewer E-cadherin positive cells.

EMT Transcriptional Reprogramming Induced by HGF and OSM.

To more fully characterize the transcriptional changes observed in this model, mRNA changes in a panel of established EMT genes were quantified (Table 3). We looked for gene expression changes greater than 3 fold after 1 and 7 days of ligand treatment to distinguish early and late changes. Other than E-cadherin, there were no changes in expression of the epithelial genes that were examined. Integrin β3 was the most consistently changed mesenchymal gene. The EMT transcription factors that were upregulated in this model were TWIST, ZEB 1 and ZEB2.

TABLE 3 Transcriptional changes to EMT genes in the CFPAC1 model. HGF OSM HGF + OSM 1 Day 7 Day 1 Day 7 Day 1 Day 7 Day CDH1 NC NC NC −3.06 NC −4.31 MMP7 NC NC NC NC NC NC MTA3 NC NC NC NC NC NC ACTN1 NC NC NC NC NC NC CDH2 NC NC NC NC NC NC ITGB3 NC 3.57 31.39 106.2 23.08 86.19 PLAUR NC NC NC NC NC NC SNAI1 NC NC NC NC NC 3.05 SNAI2 NC NC NC NC NC NC SPARC NC −5.41 NC −33 NC NC TWIST1 NC 612 NC 3.3 −3.28 675 VIM NC NC NC NC NC 5.23 ZEB1 NC NC NC 3.00 NC 3.80 ZEB2 NC NC 3.52 13.7 3.45 22.41 CFPAC1 cells were treated with ligand for 1 or 7 days. Fold changes in mRNA levels were determined by qPCR. Only changes greater than 3 fold are reported. NC indicates no change.

Phenotypic Impact of EMT in CFPAC1 Cells

To further characterize phenotypic changes associated with EMT in CFPAC1 cells, the doubling time of cells after 7 day ligand treatment was determined, since a slower growth rate is a characteristic of mesenchymal cells (Table 4). HGF treatment did not increase the doubling time of the cells, but OSM treatment did result in an increase of approximately 50%. HGF+OSM ligand treatment also increased doubling time, but the difference was not distinguishable from OSM alone.

TABLE 4 Impact of EMT on doubling time in CFPAC1 model. Doubling time (hrs) Untreated 36.0 SD 3.46 HGF 41.2 SD 5.94 OSM 54.7 SD 10.25 HGF + OSM 57.7 SD 3.11 Cells were treated for 7 days with HGF, OSM or HGF + OSM. Doubling times were calculated over the following 12 days. OSM and HGF + OSM caused an increase in doubling time, but HGF alone did not..

Impact of EMT on 3D Growth

Cells were grown suspended in a Matrigel matrix to examine the effects of EMT in a 3D environment where cells are allowed to form contacts with the extracellular matrix. After 14 days in culture with or without HGF+OSM, the cultures were fixed, and the cells immunostained for E-cadherin and vimentin, and with a nuclear stain to distinguish colony architecture (FIG. 9). Untreated colonies consisted of multiple round nodules attached together, with approximately 40% of the colonies showing hollow centers in some of the nodules. The colonies showed membrane E-cadherin staining with low levels of vimentin in some cells. When cells were grown in the presence of HGF+OSM, the colony architecture became less organized with almost all colonies showing cells growing in the center and increased vimentin staining.

In vivo Growth

It was determined whether CFPAC I cells underwent EMT in vivo. Untreated cells were implanted orthotopically and evaluated over time for EMT status by western blotting, qPCR and immunohistochemistry. The orthotopic tumors showed a gradual time-dependent decrease in E-cadherin protein and mRNA levels from week 2 to week 8 (FIG. 10A). Conversely, the tumors showed an increase in vimentin protein and mRNA from week 2 to week 8. Serial sections of the orthotopic tumors demonstrated that the tumors contain a mixture of cells with expression of E-cadherin, vimentin, or both (FIG. 10B). Groupings of cells within the tumors (arrows) may express: 1) E-cadherin but not vimentin; 2) Vimentin but not E-cadherin; or 3) both vimentin and Ecadherin. The loss of E-cadherin and gain of vimentin is a classical characteristic of EMT. However, during the EMT process cells may express both markers, perhaps representing cells which have undergone partial or incomplete EMT. Thus, CFPAC tumors consist of cells which are potentially in all three stages of EMT. Over time, more cells in the tumor may progress through the EMT process to change the overall status of the tumor as shown by the global decrease in E-cadherin and increase in vimentin when using whole tumor analysis of protein or mRNA. This CFPAC 1 orthotopic animal model may be used in the identification or characterization of new pharmacological agents that inhibit tumor cells from undergoing an epithelial to mesenchymal transition, and thus have potential value as anti-cancer agents.

Abbreviations

EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EGFR-TKI, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; EMT, epithelial-to-mesenchymal transition; MET, mesenchymal-to-epithelial transition; NSCL, non-small cell lung; NSCLC, non-small cell lung cancer; HNSCC, head and neck squamous cell carcinoma; CRC, colorectal cancer; MBC, metastatic breast cancer; Brk, Breast tumor kinase (also known as protein tyrosine kinase 6 (PTK6)); FCS, fetal calf serum; LC, liquid chromatography; MS, mass spectrometry; IGF-1, insulin-like growth factor-1; TGFα, transforming growth factor alpha; HB-EGF, heparin-binding epidermal growth factor; LPA, lysophosphatidic acid; IC₅₀, half maximal inhibitory concentration; pY, phosphotyrosine; wt, wild-type; PI3K, phosphatidyl inositol-3 kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; PDK-1, 3-Phosphoinositide-Dependent Protein Kinase 1; Akt, also known as protein kinase B, is the cellular homologue of the viral oncogene v-Akt; mTOR, mammalian target of rapamycin; 4EBP1, eukaryotic translation initiation factor-4E (mRNA cap-binding protein) Binding Protein-1, also known as PHAS-I; p70S6K, 70 kDa ribosomal protein-S6 kinase; eIF4E, eukaryotic translation initiation factor-4E (mRNA cap-binding protein); Raf, protein kinase product of Raf oncogene; MEK, ERK kinase, also known as mitogen-activated protein kinase kinase; ERK, Extracellular signal-regulated protein kinase, also known as mitogen-activated protein kinase; PTEN, “Phosphatase and Tensin homologue deleted on chromosome 10”, a phosphatidylinositol phosphate phosphatase; pPROTEIN, phospho-PROTEIN, “PROTEIN” can be any protein that can be phosphorylated, e.g. EGFR, ERK, S6 etc; PBS, Phosphate-buffered saline; TGI, tumor growth inhibition; WFI, Water for Injection; SDS, sodium dodecyl sulfate; ErbB2, “v-erb-b2 erythroblastic leukemia viral oncogene homolog 2”, also known as HER-2; ErbB3, “v-erb-b2 erythroblastic leukemia viral oncogene homolog 3”, also known as HER-3; ErbB4, “v-erb-b2 erythroblastic leukemia viral oncogene homolog 4”, also known as HER-4; FGFR, Fibroblast Growth Factor Receptor; DMSO, dimethyl sulfoxide.

Incorporation by Reference

All patents, published patent applications and other references disclosed herein are hereby expressly incorporated herein by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. 

What is claimed is:
 1. A method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising contacting a sample of cells of the epithelial tumor cell line CFPAC-1 with a test agent to be screened, contacting the sample with a single protein ligand preparation that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells, determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the increase or decrease in the expression level of a biomarker whose increase or decrease of expression level is indicative that the sample tumor cells have undergone EMT, to the increase or decrease in the expression level of the same biomarker in an identical sample of CFPAC-1 cells not contacted with the test agent, and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from any of the protein ligands that bind to and activate the receptors for OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.
 2. The method of claim 1, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from OSM; HGF; BMP7; IGF2; LIF; PAR2 agonist SLIGKV-NH₂; IL-33; PAR4 agonist AYPGKF-NH₂; CTGF; or BMP4.
 3. The method of claim 2, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in CFPAC-1 cells is selected from HGF and OSM.
 4. The method of claim 1, wherein the biomarker whose decrease of expression level is indicative that the sample tumor cells have undergone EMT is an epithelial cell biomarker.
 5. The method of claim 4, wherein the epithelial cell biomarker is E-cadherin, CDH1 promoter activity, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.
 6. The method of claim 1, wherein the biomarker whose increase of expression level is indicative that the sample tumor cells have undergone EMT is a mesenchymal cell biomarker.
 7. The method of claim 6, wherein the mesenchymal cell biomarker is vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb1, twist, FOXC2 or snail. 